Detection and Use of Prolylcarboxypeptidase

ABSTRACT

The present invention relates to weight control, control of body fat and food intake, and provides useful methods for treating, inter alia, obesity, diabetes, and conditions, diseases, and disorders relating thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is entitled to priority, pursuant to 35 U.S.C. §119(e),to U.S. provisional patent application No. 60/573,146, which was filedon May 21, 2004, which is incorporated herein in its entirety.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH OR DEVELOPMENT

This research was supported in part by U.S. Government funds (DK52581,DK61619, HL52779, HL57346, and HL65194) and the U.S. Government maytherefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

Obesity is arguably the greatest public health threat in modern Westernsociety, and it is an increasing threat throughout the world. A recentSurgeon General's report underscores the impact of obesity on humanhealth. According to the report, approximately 61% of adults in theUnited States are overweight or obese, and the prevalence of overweightchildren and adolescents has doubled in the past two decades. Theestimated economic burden of obesity to the United States alone is about$117 billion annually, and obesity is associated with an estimated300,000 deaths per year, resulting from such disorders as hypertension,dyslipidemia, diabetes, stroke, gallbladder disease, cardiovasculardisease, osteoarthritis, hypercholesterolemia, sleep apnea, respiratoryproblems, cancer, stroke and many other disorders.

In light of the health dangers attributed to obesity, many treatments,both pharmacological and non-pharmacological, have been developed tocombat this enormous problem. Pharmacological methods to control weighthave targeted a spectrum of physiological processes. Central nervoussystem (CNS) appetite suppressants interact with catecholaminergicreceptors in the brain stem or regulate available serotonin levels.Drawbacks to these agents include possible addiction and numerous sideeffects including nervousness, insomnia, drowsiness, depression, nauseaand lassitude.

Another class of pharmacologic agents for weight control promotesmalabsorption of fats and carbohydrates through inhibition of digestiveenzymes. Amylase, glycosidase and lipase inhibitors have been isolatedfrom bacterial or fungal sources, and have been used to prevent theabsorption of fats and carbohydrates in the digestive tract. A majorproblem with these agents is that it is virtually impossible to maintainphysiological levels of these inhibitors that can effectively inhibitgastrointestinal enzymes, and therefore absorption. Additionally, theuse of these inhibitors often leads to compensatory cravings for otherfoods. As an example, subjects taking a lipase inhibitor will oftenconsume more carbohydrates to compensate for the loss of fat absorptionin the diet, thereby negating any weight control benefits.

Another type of weight control agents is non-caloric, non-nutritivedietary substitutes, including saccharine, aspartame, and sucrosepolyester (a fat substitute). The sucrose substitutes, saccharine andaspartame, have been linked to hyperphagia in order to compensate forthe loss of calories from naturally occurring sucrose, and therefore maynot help control weight. Furthermore, these are only sugar substitutes,and do not impact the role of fat consumed in the diet. Sucrosepolyester is a sucrose bound to varying numbers and lengths of fattyacid chains. The size and complexity of the fatty acid chains prevent itfrom being absorbed, but also binds many fat-soluble vitamins, leadingto vitamin deficiencies. Further, sucrose polyester has been associatedwith severe and unpredictable gastrointestinal instability and fecalincontinence.

In addition to the methods directed towards controlling obesity usingpharmacological methods, a great deal of research has been conducted toelucidate the underlying genetic and biochemical mechanisms of obesity.Many human genes have been linked directly to obesity (Zhang et al.,1994, Nature 372:425-432; Deng et al., 2002, Am. J. Hum. Genet.70:1138-1151), or to obesity susceptibility (Frougel and Boutin, 2001,Exp. Biol. Med. 226:991-996). Additionally, many of the genes involvedin obesity have also been implicated in diabetes mellitus (Coleman etal., 1978, Diabetologia 14:141-148)

Monogenic human obesity syndromes have validated obesity genes expressedin the hypothalamus that involve melanocortin signaling. Melanocortins(MCs) are peptides cleaved from a common precursor, pro-opiomelanocortin(POMC). The central melanocortin system plays a critical role in thehomeostatic regulation of body weight (Cone, 1999; Fan et al., 1997;Huszar et al., 1997; Mizuno and Mobbs, 1999; Butler et al., 2000).Reduced expression of hypothalamic POMC is associated with obesitysyndromes caused by mutations in the leptin receptor (Mizuno et al.,1998; Kim et al., 2000), or other genes (tubby, Nhlh2, etc.) (Guan etal., 1998; Good et al., 1997); by hypothalamic damage (Bergen et al.,1998); and perhaps most common, by aging (Mobbs et al., 2001). Thatreduced hypothalamic POMC mRNA could contribute to the obese phenotypesin these models is suggested by the observation that mutations in thePOMC gene cause obesity in mice (Yaswen et al., 1999) and humans (Krudeet al., 1998). However, it is still unclear whether normalization ofcentral POMC tone can reverse obese phenotypes.

The prohormone POMC is processed by proteases to produce several peptidehormones including α-melanocyte stimulating hormone (α-MSH). α-MSH is amajor regulator of feeding and body weight homeostasis. α-MSH inhibitsfood intake by binding to the melanocortin receptors 3 and 4 (MC3R andMC4R) (Vaisse et al., 2000). Mutations in POMC that prevent productionof α-MSH cause monogenic human obesity (Krude et al., 1998). Mutationsin carboxypeptidase E prevent processing of many prohormones, includingPOMC, and cause obesity (Naggert et al., 1995; Duhl et al., 1994;Jackson et al., 1997; Huszar et al., 1997). Mutations in MC4R may befound in 1 to 5% of all morbidly obese individuals. Mutations in allthese genes also cause obesity syndromes in mice.

Despite the recent advances that have been made in understanding thecauses for obesity, it remains a largely intractable disorder. It wouldbe useful, therefore, to provide efficacious methods and compositionsfor the prevention, treatment and amelioration of obesity andobesity-related disorders.

BRIEF SUMMARY OF THE INVENTION

Prolylcarboxypeptidase (PRCP) is an evolutionarily conserved proteasethat specifically cleaves peptides with a penultimate proline residue(---px), including α-melanocyte stimulating hormone (α-MSH), a criticalregulator of energy homeostasis.

In humans, PRCP nucleotide polymorphism was linked to a metabolicphenotype. In mice, a chromosome 7 congenic mouse model containing only0.5 centiMorgans of BALB/c donor DNA was significantly leaner than thebackground C57BL/6Byr strain. PRCP resides in the BALB/c region. Toidentify the prolylcarboxypeptidase gene (Prcp), positional genetics wasused in a congenic mouse line containing BALC/c genome DNA on a C57BL/6Jbackground. Microarray analysis of all 97 genes in the maximal donorregion with RNA from 4 tissues revealed no significant genomewidegenotype differences, but real time PCR revealed a significant genotypeeffect on PRCP mRNA in brain, even though PRCP is in a haplotype sharedbetween BALB/c and C57BL/6J. There was a 1.7-fold greater expression inthe whole brains of congenic mice than in the background C57BL/6J mice(Table 5). This table shows differential expression measured with 4separate assays for Prcp and normalized for expression of 4 separatecontrol genes.

In situ hybridization shows that the PRCP gene is expressed inhypothalamic regions associated with feeding regulation, includingarcuate (ARC) nucleus and ventral medial hypothalamus (VMH). Thislocation suggests a close association between brain PRCP andα-MSH-producing melanocortin cells. PRCP immunopositive neurons in thehypothalamus of non-human primate were also detected confirming anevolutionarily conserved system.

The anorectic neuropeptide α-MSH, the effecter of POMC feeding neuronsinhibiting food intake through melanocortin receptor 4 (MC4), is apotential substrate of PRCP because α-MSH contains a penultimate prolineresidue: “SYSMEHFRWGKPV” (SEQ ID NO: 1). It is shown that inhibiting.PRCP should increase anorexigenic tone and suppress food intake andweight gain. In contrast to full length α-MSH, the degradation productof α-MSH catalyzed by PRCP has neither neuromodulatory nor anorexigeniceffects.

Conversely, inhibition of PRCP activity by protease inhibitors resultsin suppressed food intake in wild type as well as in genetically obese(ob/ob) mice. Administration of a selective PRCP inhibitor, t-butylcarbamate (BOC)-prolyl prolinal (B-PP), reduced food intake regardlessof central or peripheral administration. Food intake reduction wasevident even in genetically obese, ob/ob mice. Administration of anotherPRCP inhibitor, N-benzyloxycarbonyl-prolyl-prolinal (Z-PP), also reducedfood intake in after peripheral administration to ob/ob mice. In any ofthe cases described, food intake returned to control levels aftercessation of drug administration, which was not followed by reboundfeeding.

Inactivation of Prcp gene in mice unmasked food intake inhibition byperipheral α-MSH administration which was not seen in wild typelittermates. These observations reveal PRCP as a target for furtherobesity drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a graph depicting the catalytic activity of PRCP on α-MSH,MSH₁₋₁₂ and angiotensinogin II (AgII).

Panel A: Increasing concentration of α-MSH, MSH1-12, or AgII (0.001-1mM) were incubated with 8 nM of recombinant PRCP51 at 37° C. inmicrotiter plate cuvette wells with preabsorbed HK and containing 20 nMPK. The liberation of paranitroanilide (pNA) from the S2302 by theformed plasma kallikrein in the presence of the peptide was measured at405 nm and the results are expressed as residual formed plasmakallikrein activity. The data represent the mean±SEM of threeindependent measurements.

Panel B: Real time PCR of DNA from mouse tail or cells for lacZ. Theprobes that were used for these studies are indicated in Table 4. Thecurves for lacZ DNA shown are for the wild type (ES) or PRCP genetrapped PRCP cell line (KST302), respectively. The curves for PRCP^(−/−)or PRCP^(+/+) indicates PCR of tail DNA from presumed KO or littermatewild type mouse, respectively. The figure is a representative real timePCR on a single mouse or cell DNA sample.

Panel C: RNA from kidney from PRCP^(−/−) or PRCP^(+/+) mice or ES orKST302 cells were subjected to reverse transcription, real time PCRusing probes described in Table 4. Primers for Kidney RNA real time PCRare described in Table 4. Amplification plots show the fluorescenceplotted against cycle number for the reverse transcribed RNA around theinsertional mutation site using sense primers to exon 5 of PRCP andantisense primers to the TM region of pGT1.8 vector and a probe commonto the contiguous sequence of exon 5 with the splicing acceptor (SA)region of pGT1.8TM vector (Table 4). The figure is a representativestudy of the mouse kidney RNA from a wild type and KO animal shown inPanel B.

Panel D: RNA from kidney from PRCP^(−/−) or PRCP^(+/+) mice weresubjected to reverse transcription, real time PCR using probes describedin Table 4. Amplification plots show the fluorescence of a probe thatspans the 3′ region of exon 5 and splice acceptor region of the vectorof the cDNA using various combinations of sense primers specific toexons 1 or 5 of PRCP and antisense primers to the TM region of pGT1.8vector or exons 8, 9, or 10 of PRCP.

FIG. 2 is an image of In situ hybridization showing that the PRCP geneis expressed in hypothalamic regions.

Panel A: Dark blue beta galactosidase labeling representing LacZexpression in the place of PRCP gene in cells of the hypothalamus. Mostlabeled cells are in an area in the vicinity of the dorsomedialhypothalamic nucleus (DMH), perifornical region (pf) lateralhypothalamus (LH) and zona incerta (ZI). Few labeled cells are alsovisible in the arcuate nucleus (ARC) of the mediobasal hypothalamus.Fornix, III: third ventricle.

Panel B: Corresponding to the LacZ expression shown on panel A, in situhybridization for PRCP in wild type animals resulted in labeled cells(black dots representing digoxigenin-labeling of anti sense mRNA probe)in the DMH, pf, LH and ZI with few labeled cells also present in theARC.

Panels C-F: Double labeling for beta galactosidase (LacZ) and melaninconcentrating hormone (MCH), hypocretin (Hcrt) and pro-opiomelanocortin(POMC) revealed extensive coexpression of PRCP and MCH in the lateralhypothalamus-perifornical region (C) and zona incerta (D), and PRCP andHcrt in the lateral hypothalamus-perifornical region (E). Few cellsexpressing POMC were also found to express LacZ (representing PRCP) inthe arcuate nucleus. Red arrows indicate double labeled cells, blackarrows point to single labeled LacZ expressing cells and blackarrowheads indicate single labeled MCH, Hcrt or POMC neurons.

FIG. 3A is a graph showing the effect of intracerebral ventrical (ICV)injection of 2.5 mg of α-MSH and α-MSH₁₋₁₂ on food intake compared tosaline control.

FIG. 3B shows the electrophysiologic results of α-MSH and α-MSH₁₋₁₂ onGFP POMC neurons of the arcuate nucleus of the hypothalamus.

FIG. 3C is a graph showing the effect of intracerebral ventrical (ICV)administration of vehicle and 0.9 mg B-PP on food intake of fasted rats.

FIG. 3D is a graph showing the percentage of food intake in ob/ob miceinjected intraperitoneally with vehicle (open bars) and 400 μg of B-PP(black bars). *P<0.05

FIG. 3E is a graph showing the percentage of food intake in ob/ob miceinjected intraperitoneally with vehicle (open bars) and 400 μg of Z-PP(black bars). *P<0.05

FIG. 4A shows the percentage of food intake after intraperitoneal (IP)injection of 200 nmol of α-MSH in wild type (WT) or PRCP KO micecompared to saline control animals.

FIG. 4B shows food intake in grams after intraperitoneal (IP) injectionof 200 nmol of α-MSH in wild type (WT) or PRCP KO mice compared tosaline control animals. * P<0.05

DETAILED DESCRIPTION OF THE INVENTION

The present invention is partly based on the discovery that inhibitionof PRCP decreases food intake, body fat, and affects physical activityand metabolic rate. These unexpected results demonstrate that inhibitionof PRCP is a treatment for a disease, disorder or condition mediated byfood intake, and the like. Such a disease, disorder or conditionincludes, but is not limited to, obesity, hypertension, dyslipidemia,diabetes, stroke, gallbladder disease, cardiovascular disease,osteoarthritis, rheumatoid arthritis, hypercholesterolemia, stableangina, unstable angina, arthrosclerosis, sleep apnea, respiratoryproblems, cancer, stroke, and the like.

The present invention also includes methods of identifying additionalcompounds that inhibit PRCP for affecting a disease, disorder orcondition mediated by increased body fat, decreased physical activities,decreased metabolic activity, and the like.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“Prolylcarboxypeptidase” and “PRCP” as used herein refer to anevolutionarily conserved protease that specifically cleaves peptideswith a penultimate proline residue (---px), including α-melanocytestimulating hormone (α-MSH), a critical regulator of energy homeostasis.PRCP is also known by various other names by those skilled in the artincluding, but not limited to prolylcarboxypeptidase Pro-X (PRCP-Pro-X),angiotensinase C, Pro-x carboxypeptidase, Lysosomal Pro-Xcarboxypeptidase precursor, Proline carboxypeptidase and Lysosomalcarboxypeptidase C.

“PRCP activity” is used herein to refer the function of theprolylcarboxypeptidase, including, but not limited to, cleaving thepeptides with a penultimate proline residue (---px, wherein “---”designates the preceding amino acids of the protein, “p” designates aproline residue, and “x” designates any amino acid) resulting a changein the peptides function, and the like.

“PRCP inhibiting” is used herein to refer to detectably decreasing theactivity, function and/or expression of PRCP.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for administering the inhibitor of PRCP of the inventionto a mammal.

The term “inhibitor” is used herein to refer to a composition of matterwhich when administered to a mammal such as a human, inhibits abiological activity attributable to the level or presence of anendogenous compound in the mammal.

The term “weight loss” is used herein to refer to a detectable decreaseof body mass in an animal compared to the mass of the animal at aprevious time.

As used herein, “an effective amount of an PRCP inhibitor” means anamount of a compound which effects a detectable decrease in theexpression, activity, or both, of PRCP in a cell, an animal, or both,compared with the level of expression, activity, or both, of PRCP in anotherwise identical cell or animal to which the compound is notadministered, or in the same cell or animal prior to administration ofthe compound.

As used herein, the term “antisense nucleic acid molecule” means anucleic acid polymer, at least a portion of which is complementary to anucleic acid, which is present in a normal cell or in an affected cell.The antisense nucleic acid molecules of the invention preferablycomprise between about ten and about one hundred nucleotides. Mostpreferably, the antisense nucleic acid molecules comprise between aboutfifteen and about fifty nucleotides. The antisense oligonucleotides ofthe invention include, but are not limited to, phosphorothioateoligonucleotides and other modifications of oligonucleotides. Methodsfor synthesizing oligonucleotides, phosphorothioate oligonucleotides,and otherwise modified oligonucleotides are well known in the art (U.S.Pat. No. 5,034,506; Nielsen et al., 1991, Science 254: 1497).

As used herein, the term “obesity-related disorder” refers to anycondition where the accumulation of excess fat is a risk factor fordeveloping health complications. Over time, weight loss in obeseindividuals may reduce a number of health risks. Studies looking at theeffects of weight-loss medication treatment on obesity-related healthrisks have found that some agents lower blood pressure, bloodcholesterol, and triglycerides (fats) and decrease insulin resistance(the body's inability to use blood sugar) via a reduction in weight.

For purposes of the present invention, obesity-related disordersinclude, but are not limited to, hypertension, dyslipidemia, diabetes,stroke, gallbladder disease, cardiovascular disease, osteoarthritis,rheumatoid arthritis, hypercholesterolemia, stable angina, unstableangina, sleep apnea, respiratory problems, cancer, strokehyerinsulinemia, Syndrome X, hypercholesterolemia, hyperlipoproteinemia,hypertriglyceridemia, atherosclerosis, diabetic renal disease and manyother disorders. Thus, the reduction of excess fat by the methods andcompositions of the present invention also helps to prevent or to treatany health complication arising from the condition of having the excessfat.

By the term “specifically binds” as used herein, is meant an antibodywhich recognizes and binds PRCP, but does not substantially recognize orbind other molecules in a sample.

“Physical activity” is used herein to refer to any detectable movement,action, and/or alertness in an animal.

“Affecting physical activity” in an animal encompasses mediating adetectably higher or lower level of movement, action, and/or alertnessin the animal, whereas decreasing physical activity in an animal isachieving a lower level of movement, action, and/or alertness.

“Metabolic rate” is used herein to refer to the use of calories oranother energy source in an animal as assessed over a period of time.Hence, an increased metabolic rate is a higher level of the use ofcalories or another energy source by an animal over a period of timecompared with the level of use of calories by an otherwise identicalanimal over the same period of time under substantially similar oridentical conditions.

Description

I. Methods

A. Method of Treating and/or Preventing a Disease Disorder or Condition

The present invention is based, in part, on the novel discovery thatPRCP plays a significant role in weight control in a mammal. Asdemonstrated by the data disclosed herein, inhibition of PRCP can induceweight loss, decrease body fat, affect appetite, decrease food intake,both treat and prevent obesity. The data disclosed here demonstrate thatPRCP is involved in the signal transduction pathway for food intake andweight control. Thereby, the present invention discloses methods totreat various diseases relating to food intake and weight control,including, but not limited to, obesity, hypertension, dyslipidemia,diabetes, stroke, gallbladder disease, cardiovascular disease,osteoarthritis, hypercholesterolemia, sleep apnea, respiratory problems,cancer, stroke and many other disorders.

The present invention includes a method of inducing weight loss in ananimal. This is because, as demonstrated by the data disclosed elsewhereherein, inhibition of PRCP, whether its expression, biological activity,or both, is inhibited, mediates a variety of physiological responses,including, but not limited to, decreased body fat, decreased foodintake, increased physical activities, and increased metabolic rate.Thus, inhibiting PRCP can be used to treat a wide variety of diseases,disorders or conditions where decreasing body fat and food intake wouldprovide a therapeutic benefit to an animal. Accordingly, one skilled inthe art would appreciate, based upon the disclosure provided herein,that inhibiting PRCP provides an important, novel therapeutic fortreatment of, inter alia, obesity, hypertension, dyslipidemia, diabetes,stroke, gallbladder disease, cardiovascular disease, osteoarthritis,hypercholesterolemia, sleep apnea, respiratory problems, cancer, strokeand many other disorders, and the like. More particularly, PRCP can beinhibited thereby mediating weight loss in an animal.

The skilled artisan would appreciate, based upon the disclosure providedherein, that an animal encompasses a bird, a fish, and a mammal, wherethe mammal includes, but is not limited to, a rodent, a cow, a pig, asheep, a buffalo, a beefalo (a cow/buffalo hybrid animal), a bison, adeer, a goat, and a human. One skilled in the art, armed with theteachings provided herein, would appreciate that the animal comprisesPRCP, which can be inhibited.

An inhibitor of PRCP is administered to the animal thereby decreasingPRCP and providing a therapeutic benefit. The skilled artisan wouldappreciate, based upon the disclosure provided herein, that PRCP can beinhibited using a wide plethora of techniques well-known in the art orto be developed in the future. That is, the invention encompassesinhibiting PRCP expression, e.g., inhibition of transcription and/ortranslation. This is because, as demonstrated by the data disclosedelsewhere herein, knocking-out the nucleic acid encoding PRCP, such thatthe nucleic acid was not transcribed or translated, mediated a varietyof effects, including, but not limited to, decreased food intake, lossof weight, and a decrease in body fat. Thus, inhibiting PRCP includes,but is not limited to, inhibiting translation and/or transcription of anucleic acid encoding the protein.

Further, the routineer would understand, based upon the disclosureprovided elsewhere herein, that inhibition of PRCP includes, but is notlimited to, inhibiting the biological activity of the molecule. This isbecause, as the data disclosed elsewhere herein demonstrate, inhibitionof PRCP activity using a PRCP inhibitor (e.g., B-PP) mediated a decreasein food intake and body fat. These data indicate that inhibition of PRCPactivity provides a therapeutic benefit for treatment of a disease, suchas, but not limited to, obesity, or diabetes.

The skilled artisan would understand that an inhibitor of PRCPencompasses, but is not limited to, an inhibitor of a carboxypeptidase,including, but not limited to, t-butyl carbamate (BOC)-prolyl prolinal(B-PP), N-benzyloxycarbonyl-prolyl-prolinal (Z-PP), diisopropylfluorophosphate, PMSF, antipain, leupeptin, corn trypsin, and mercuricchloride (high concentrations).

One skilled in the art, based upon the disclosure provided herein, wouldappreciate that PRCP inhibition can be mediated by using, inter alia, anantibody, an antisense nucleic acid, a siRNA, an aptamer, a ribozyme, asmall molecule, a peptidomimetic, and a chemical compound, either knownor to be developed, which inhibits PRCP expression, activity, or both.That is, the invention encompasses using a PRCP inhibiting compound suchas, but not limited to, B-PP and Z-PP. This is because, as isdemonstrated by the data disclosed elsewhere herein, inhibition of PRCPexpression and/or activity leads to reduced food intake, reduced bodyweight, and the like.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods described herein, recognize that PRCPinhibitors include such inhibitors as discovered in the future, as couldbe established by well known criteria in the art of pharmacology, andthose identified in light of the physiological results of inhibition ofPRCP as described in detail herein. Therefore, the present invention isnot limited in any way to the PRCP inhibitors described herein, butincludes those PRCP inhibitors, and other carboxypeptidase inhibitors,that inhibit, inter alia, PRCP, as are discovered in the future.

Methods of obtaining and generating carboxypeptidase inhibitors are wellknown to those of ordinary skill in the art. For instance, acarboxypeptidase inhibitor can be isolated from a naturally occurringsource. Likewise, a carboxypeptidase inhibitor can be readilysynthesized chemically.

An inhibitor of PRCP may be an antibody that specifically binds to PRCPthereby inhibiting the action of PRCP. Antibodies that specifically bindto PRCP are well known to those of ordinary skill in the art(Shariat-Madar et al., 2002, 2004), may be purchased commercially, orcan be produced using standard methods (Harlow et al., 1988, Antibodies:A Laboratory Manual, Cold Spring Harbor, N.Y.).

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

One of skill in the art will appreciate that an antibody can beadministered as a protein, a nucleic acid construct encoding a protein,or both. Numerous vectors and other compositions and methods are wellknown for administering a protein or a nucleic acid construct encoding aprotein to cells or tissues. Therefore, the invention includes a methodof administering an antibody or nucleic acid encoding an antibody(synthetic antibody) that is specific for PRCP (Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York; Ausubel et al., 1997, Current Protocols in Molecular Biology,John Wiley & Sons, New York).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

One skilled in the art would understand, based upon the disclosureprovided herein, that an antibody can be administered such that itinhibits the function of PRCP present in a membrane. Moreover, theinvention encompasses administering an antibody that specifically bindswith PRCP, or a nucleic acid encoding the antibody, wherein the moleculefurther comprises an intracellular retention sequence such that antibodybinds with the PRCP and prevents its expression in a membrane. Suchantibodies, frequently referred to as “intrabodies”, are well known inthe art and are described in, for example, Marasco et al. (U.S. Pat. No.6,004,490) and Beerli et al. (1996, Breast Cancer Research and Treatment38:11-17). Thus, the invention encompasses methods comprising inhibitingPRCP where the PRCP is present in a cell membrane, as well as methods ofinhibiting PRCP comprising inhibiting the PRCP being present in the cellmembrane, and such methods as become known in the future.

As noted previously elsewhere herein, the present encompasses inhibitingPRCP by inhibiting expression of a nucleic acid encoding PRCP. Methodsfor inhibiting the expression of a gene are well known to those ofordinary skill in the art, and include the use of antisense nucleic acidmolecules, ribozymes, interference RNA, or aptamers.

Antisense nucleic acid molecules are DNA or RNA molecules that arecomplementary to some portion of an mRNA molecule. When present in acell, antisense nucleic acids hybridize to an existing mRNA molecule andinhibit translation into a gene product. Inhibiting the expression of agene using an antisense nucleic acid molecule is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods to expressan antisense nucleic acid molecule in a cell (Inoue, 1993, U.S. Pat. No.5,190,931).

Antisense nucleic acid molecules can be synthesized and provided to thecell by way of methods well known to those of ordinary skill in the art.Antisense nucleic acid molecules can be synthesized, for example, to bebetween about 10 and about 100, more preferably between about 15 andabout 50 nucleotides long. The synthesis of nucleic acid molecules iswell known in the art. The antisense nucleic acid molecule can also bemodified to improve biological activity in comparison to unmodifiedantisense nucleic acid molecules (Tullis, 1991, U.S. Pat. No.5,023,243).

Inhibition of expression of PRCP can also be achieved by using aribozyme. Using ribozymes for inhibiting gene expression is well knownto those of ordinary skill in the art (Cech et al., 1992, J. Biol. Chem.267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman et al.,1992, U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA moleculeswith the ability to cleave other single-stranded RNA molecules.Ribozymes are known to be sequence specific, and can therefore bemodified to recognize a specific nucleotide sequence (Cech, 1988, J.Amer. Med. Assn. 260:3030), allowing the selective cleavage of specificmRNA molecules. Given the nucleotide sequence of PRCP is well known inthe art, one of ordinary skill in the art can synthesize an antisensepolynucleotide or ribozyme without undue experimentation, provided withthe disclosure and references incorporated herein.

Another way of inhibition of expression of PRCP is by using RNAinterference (RNAi). RNAi is a phenomenon in which the introduction ofdouble-stranded RNA (dsRNA) into a diverse range of organisms and celltypes causes degradation of the complementary mRNA. In the cell, longdsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs,or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequentlyassemble with protein components into an RNA-induced silencing complex(RISC), unwinding in the process. Activated RISC then binds tocomplementary transcript by base pairing interactions between the siRNAantisense strand and the mRNA. The bound mRNA is cleaved and sequencespecific degradation of mRNA results in gene silencing. For example, thepresent invention encompasses methods of inhibiting expression of a Prcpgene in a cell in an animal comprising providing at least oneribonucleic acid (RNA) to the cell in an amount sufficient to inhibitthe expression of a Prcp gene, wherein the RNA comprises or forms adouble-stranded structure containing a first strand consistingessentially of a ribonucleotide sequence which corresponds to anucleotide sequence of a Prcp gene and a second strand consistingessentially of a ribonucleotide sequence which is complementary to thenucleotide sequence of the Prcp gene, wherein the first and the secondribonucleotide sequences are complementary sequences that hybridize toeach other to form said double-stranded structure, and the RNAcomprising the double-stranded structure inhibits expression of a Prcpgene. The RNA can be provided to the cell by contacting the cell withthe RNA. Alternatively, the RNA can be provided to the cell bysynthesizing the RNA in the cell. For further information see, forexample, U.S. Pat. No. 6,506,559; WO 00/01846; WO 00/44914; WO 00/44895;WO 00/63364; WO 01/68836; WO 01/75164; WO 02/44321; WO 03/010180; USPatent Application Publication No. 2003/0148519; WO 03/068797; US PatentApplication Publication No. 2003/0203868; WO 99/61631; Fire et al.,Nature (1998) 391(19):306-311; Timmons et al., Nature (1998) 395:854;Montgomery et al., TIG (1998) 14(7):255-258; David R. Engelke, Ed., RNAInterference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press (2003);Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold SpringHarbor Laboratory Press (2003); and Krishnarao Appasani, Ed., RNAInterference Technology, Cambridge University Press (2005), each one ofwhich is specifically and completely incorporated by reference herein.Therefore, the present invention also includes methods of silencing thegene encoding PRCP by using RNAi technology.

Inhibition of PRCP can also be achieved by using an aptamer. Aptamersare nucleic acid molecules having specific binding affinity to moleculesthrough interactions other than classic Watson-Crick base pairing.Aptamers, like peptides generated by phage display or monoclonalantibodies (MAbs), are capable of specifically binding to selectedtargets and, through binding, block their targets' ability to function.Created by an in vitro selection process from pools of random sequenceoligonucleotides, aptamers have been generated for over 100 proteinsincluding growth factors, transcription factors, enzymes,immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size(30-45 nucleotides), binds its target with sub-nanomolar affinity, anddiscriminates against closely related targets (e.g., will typically notbind other proteins from the same gene family). A series of structuralstudies have shown that aptamers are capable of using the same types ofbinding interactions (e.g., hydrogen bonding, electrostaticcomplementarity, hydrophobic contacts, and steric exclusion) that driveaffinity and specificity in antibody-antigen complexes. Aptamers have anumber of desirable characteristics for use as therapeutics (anddiagnostics) including high specificity and affinity, biologicalefficacy, and excellent pharmacokinetic properties. In addition, theyoffer specific competitive advantages over antibodies and other proteinbiologics, for example: Speed and control. Aptamers are produced by anentirely in vitro process, allowing for the rapid generation of initialtherapeutic leads. In vitro selection allows the specificity andaffinity of the aptamer to be tightly controlled and allows thegeneration of leads against both toxic and non-immunogenic targets.Given the nucleotide sequence of PRCP is well known in the art, one ofordinary skill in the art can synthesize a PRCP-specific aptamer thattargets and blocks the function of PRCP.

The inhibitors of PRCP or of PRCP gene expression may be administeredsingly or in any combination thereof. Further, inhibitors of PRCP may beadministered singly or in any combination thereof in a temporal sense,in that they may be administered simultaneously, before, and/or aftereach other. One of ordinary skill in the art will appreciate the use ofPRCP inhibitors or inhibitors of PRCP gene expression to affect weightloss, a treatment of obesity, and will use the inhibitors detailedherein alone or in any combination to effect such results.

Thus, the invention encompasses a method for inducing weight loss wherean inhibitor of PRCP expression, activity, or both, is administered toan animal, thereby effecting a decreased food intake and therebymediating weight in loss by the animal.

Similarly, the present invention encompasses a method for decreasingbody fat in an animal. The method comprises administering a PRCPinhibiting amount of a PRCP inhibitor to the animal. This is because, asmore fully set forth previously elsewhere herein, the data disclosedherein demonstrate that inhibition of PRCP (expression, activity, orboth) mediates a decrease in body fat, thereby decreasing body fat inthe animal.

One skilled in the art would appreciate that having a leaner animal,i.e., an animal with decreased body fat, would provide a benefit in thatsuch an animal would be useful in providing a leaner source of meathaving a decreased amount of fat. Such animal includes, but is notlimited to, a bird, a fish, a rodent, a cow, a pig, a sheep, a buffalo,a beefalo, a bison, a deer, and a goat. Basically, the inventionincludes producing an animal comprising a decreased body fat contentwhere the animal is used for human consumption.

The invention includes a method for decreasing food intake in a mammal.Basically, the method comprises administering a PRCP inhibiting amountof a PRCP inhibitor to a mammal. This, in turn, decreases food intake inthe mammal. This is because, as disclosed elsewhere herein, the datademonstrate that inhibition of PRCP mediates a decrease in the foodintake by a mammal compared to an otherwise identical animal to whichthe inhibitor is not administered or compared with the food intake bythe same animal before administration of the PRCP inhibitor. Thus, theinvention encompasses a method of decreasing food intake by inhibitingPRCP.

The amount, dosing regimen, and route of administration for inhibitingPRCP in an animal can be readily determined by one skilled in the artand would depend on well-known factors, including, but not limited to,the age and condition of the animal, the weight and body fat content ofthe animal, and the desired weight and/or body fat content of theanimal. The dosage and route of administration can be easily determinedas exemplified elsewhere herein using art-recognized models of obesityand diabetes, and therefore, the skilled artisan would understand, basedupon the disclosure provided herein, precisely how to inhibit PRCP topractice the methods of the invention.

Similarly, the invention includes a method for affecting appetite in amammal. Once again, the method comprises administering a PRCP inhibitingamount of a PRCP inhibitor to a mammal, thereby affecting appetite inthe mammal. This is because, as discussed previously elsewhere herein,the data disclosed demonstrate that inhibition of PRCP decreasesappetite, as indicated by decreased food intake, in a mammal. Thismethod is useful for providing a therapeutic benefit where the mammal isin need of reducing their appetite, such as when, for instance, themammal is obese and/or suffers from diabetes or any other diseasemediated by, or associated with, increased weight.

The invention encompasses a method for treating obesity in a mammal. Themethod comprises administering a PRCP inhibiting amount of a PRCPinhibitor to a mammal. As demonstrated by the data disclosed herein,including, but not limited to, data demonstrating weight loss by obesemice using an art-recognized model of obesity, by inhibiting PRCP in themice, inhibiting PRCP inhibits food intake and decreases weight, therebytreating obesity in the mammal.

The present invention includes a method for preventing obesity in amammal. The method comprises administering a PRCP inhibiting amount of aPRCP inhibitor to a mammal. This is because the data disclosed elsewhereherein demonstrate that inhibiting PRCP not only treats obesity, butalso actually prevents obesity in an art-recognized model of obesity.Thus, the skilled artisan would appreciate, based on the teachingsprovided herein, that the invention includes, but is not limited to, amethod for preventing obesity in a mammal.

The present invention further includes a method for affecting physicalactivity in an animal. The method comprises administering a PRCPinhibiting amount of a PRCP inhibitor to an animal thereby affectingphysical activity. This is because, as demonstrated by the datadisclosed elsewhere herein, of PRCP affects a number of physiologicalresponses, including, but not limited to increased physical activities.Thus, inhibiting PRCP can be used to treat a wide variety of disorders,diseases, and/or conditions where decreasing body fat, affectingactivity, decreasing food intake, would provide a therapeutic benefit tomammal. Therefore, the skilled artisan will appreciate, based on thedisclosure provided herein, that surprisingly, inhibition of PRCPprovides a novel method for affecting the physical activity of ananimal.

One skilled in the art would understand, based upon the disclosureprovided herein, that affecting PRCP encompasses both inhibiting andincreasing PRCP expression, activity, or both. This is because theskilled artisan would appreciate, once armed with the teachings providedherein, that inhibiting PRCP increases physical activity such thatincreasing PRCP activity and/or expression can decrease physicalactivity in an animal. Thus, the present invention includes increasingand decreasing PRCP activity and/or expression thereby either increasingor decreasing physical activity in an animal. Preferably, PRCP activityand/or expression is inhibited thereby increasing physical activity inan animal.

The skilled artisan would further appreciate, once armed with theteachings provided herein, that a method of increasing physical activitycan be used to treat a disease or condition mediated by, or associatedwith, decreased physical activity. Such diseases or conditions include,but are not limited to, depression, narcolepsy, fatigue, and the like.

The present invention includes a method of affecting metabolic rate inan animal. This is because, as demonstrated by the data disclosedelsewhere herein, inhibition of PRCP affects a variety of physiologicalprocesses, including, but not limited to, decreased food intake,increased physical activities, and increased metabolic rate. Thus, thedata disclosed herein demonstrate that inhibiting PRCP can affectmetabolic rate in an animal. Accordingly, one skilled in the art wouldappreciate, based upon the disclosure provided herein, that inhibitingPRCP provides an important, novel therapeutic for treatment of, interalia, obesity and diabetes, atherosclerosis, diabetic renal disease, andthe like, where affecting the metabolic rate mediates a therapeuticbenefit. More particularly, PRCP can be inhibited thereby affectingmetabolic rate in an animal; therefore, inhibition of PRCP can be usedto treat, inter alia, obesity and other diseases, disorders andconditions related to decreased metabolic rate.

However, the present invention is not limited to increasing themetabolic rate. Rather, the invention encompasses methods for decreasingthe metabolic rate where such decrease would provide a benefit. This isbecause the data disclosed herein demonstrate that inhibiting PRCPmediates an increase in metabolic rate such that increasing PRCP candecrease metabolic rate. Decreasing the metabolic rate in an animal canbe used, for instance, where weight gain and/or increased body fatcontent in the animal is desired or would provide a therapeutic benefit.Conditions and diseases where decreased metabolic rate, increased bodyfat, and the like, is desirable would be readily apparent to one skilledin the art based upon the disclosure provided herein.

The invention includes a method for increasing physical activity and/ormetabolic rate in a mammal. The skilled artisan would appreciate, basedupon the teachings provided herein, that PRCP inhibitors can treatphysical activity-related diseases or disorders such as narcolepsy,depression, and the like, and that the PRCP inhibitors can be used totreat metabolism-related disease such as obesity.

The invention also encompasses the use of a pharmaceutical compositioncomprising an appropriate PRCP inhibitor to practice the methods of theinvention, where the composition comprises an appropriate PRCPinhibitor, which can be used in an amount sufficient to inhibit PRCPthereby producing a therapeutic effect, and a pharmaceuticallyacceptable carrier.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 1 ng/kg/day and 100mg/kg/day.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, and topical or other similarformulations. In addition to the appropriate PRCP inhibitor, suchpharmaceutical compositions may contain pharmaceutically acceptablecarriers and other ingredients known to enhance and facilitate drugadministration. Other possible formulations, such as nanoparticles,liposomes, resealed erythrocytes, and immunologically based systems mayalso be used to administer an appropriate PRCP inhibitor according tothe methods of the invention.

Compounds, which are identified using any of the methods describedherein, may be formulated and administered to a mammal for treatment ofthe diseases disclosed herein are now described.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesdisclosed herein as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions, are suitablefor ethical administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats and dogs, andbirds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide pharmaceutically elegant andpalatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Morepreferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

B. A Method of Identifying a Useful Compound

The invention encompasses a method for identifying a compound thatinhibits PRCP. One skilled in the art would appreciate, based upon thedisclosure provided herein, that assessing the expression, activity, orboth, of PRCP can be performed by assessing, inter alia, the levels ofPRCP in an animal, and the like, when compared to the same parameter inan otherwise identical animal not treated with the compound. One skilledin the art would understand that such compounds can be useful for,decreasing food intake, decreasing percent body fat, increasing theresting metabolic activity, and initiating weight loss. This is becausethe data disclosed elsewhere herein demonstrate, for the first time,that inhibiting PRCP (expression, activity, or both) mediates a varietyof beneficial effects, including, but not limited to, decreased foodintake, decreased body fat, weight loss, decreased caloric intake,increased resting metabolic activity, and initiating weight loss. Thisis because the present invention discloses, for the first time, thatPRCP expression is associated with, or mediates, such diseases,conditions and disorders. Accordingly, the data disclosed elsewheresuggest that inhibiting of PRCP can provides a useful therapeutic forthose diseases, disorders or conditions.

The method comprises administering to a mammal a compound and comparingthe level of PRCP activity in the mammal before and after administrationof the compound. The routineer would understand, based on the disclosureprovided herein, that a lower level of PRCP activity in the mammal afteradministration of the compound compared with the level of PRCP activitybefore administration of the compound indicates that the compound isuseful for treating a disorder, condition or disease related to obesity,including, but not limited to, hypertension, dyslipidemia, diabetes,stroke, gallbladder disease, cardiovascular disease, osteoarthritis,rheumatoid arthritis, hypercholesterolemia, stable angina, unstableangina, sleep apnea, respiratory problems, cancer, strokehyerinsulinemia, Syndrome X, hypercholesterolemia, hyperlipoproteinemia,hypertriglyceridemia, atherosclerosis, diabetic renal disease, and thelike.

This is because, as stated previously elsewhere herein, it has beendiscovered that inhibiting PRCP activity in an animal treats a diseaseassociated with PRCP expression and/or activity. The skilled artisanwould also appreciate, in view of the disclosure provided herein, thatassays to determine the level of PRCP activity and/or expression in amammal include those well known in the art, or those to be developed inthe future, all of which can be used to assess the level of PRCPactivity in a mammal before and after administration of the compound.The skilled artisan would further appreciate that PRCP activity, asdisclosed elsewhere herein, includes an association with restingmetabolism rate, caloric consumption, and the like. Further, theinvention encompasses a compound identified using this method.

The invention further includes another method of identifying a compoundthat useful for treating obesity. The method comprises assessing thelevel of PRCP activity in a mammal after administration of a compoundcompared to a standard baseline previously established for thatparticular species of mammal before administration of the compound.Methods of assaying PRCP activity are disclosed elsewhere herein, butcan include assessing caloric intake, resting metabolic activity, amountof food intake, and weight loss. A decrease in PRCP activity in a mammalafter administration of a compound compared to the baseline level ofPRCP activity determined for the that particular species of mammalbefore administration of a compound can indicate that a compound isuseful in inhibiting the activity of PRCP, and is therefore useful inthe treatment of obesity. This is because, as discussed previouslyelsewhere herein, inhibition of PRCP mediates a decrease in, inter alia,food intake and weight, thereby treating obesity, such that it would beappreciated by the skilled artisan, based on the disclosure providedherein, that a compound that inhibits PRCP can be used to treat obesity.

The invention encompasses a method of identifying a compound that isuseful for treating obesity. The method comprises assessing the level ofPRCP activity in a mammal after administration of a compound compared tothe level of PRCP in the same mammal prior to the administration of thecompound, or to the level of PRCP activity in an otherwise identicalmammal to which the compound is not administered. A decrease in PRCPactivity in a mammal after administration of a compound compared to thelevel of PRCP activity determined for the mammal prior to theadministration of the compound, or compared with the level of PRCPactivity in the otherwise identical mammal to which the compound is notadministered, is an indication that the compound is useful for thetreatment of obesity. This is because, as more fully discussed elsewhereherein, the data disclosed herein demonstrate, for the first time, thatinhibiting the activity of PRCP mediates a decrease in, inter alia, foodintake and weight, thereby treating obesity, such that it would beappreciated by the skilled artisan, based on the disclosure providedherein, that a compound that inhibits PRCP can be used to treat obesity.

The invention encompasses a method of identifying a compound that isuseful for treating obesity. The method comprises assessing the level ofPRCP activity in a cell after the cell is contacted with a compoundcompared with the level of PRCP in an otherwise identical cell which isnot contacted with the compound. A decrease in PRCP activity in the cellcontacted with the compound compared with the level of PRCP activity inthe otherwise identical cell not contacted with the compound is anindication that the compound is useful for the treatment of obesity.This is because, as more fully discussed elsewhere herein, the datadisclosed herein demonstrate, for the first time, that inhibiting theactivity of PRCP mediates a decrease in, inter alia, food intake andweight, thereby treating obesity, such that it would be appreciated bythe skilled artisan, based on the disclosure provided herein, that acompound that inhibits PRCP can be used to treat obesity.

The skilled artisan would appreciate, based upon the disclosure providedherein, that the cell used to assess the level of PRCP activity due tothe cell being contacted with a compound can be selected from the groupconsisting of a liver cell, a brain cell, a gonadal white adipose cell,a gastrocnemius muscle cell, and the like.

The skilled artisan would appreciate, based upon the disclosure providedherein, that for each of the methods of identifying a compound ofinterest, the invention encompasses any compound identified thereby.

II. Transgenic Animals

The skilled artisan will appreciate, as disclosed elsewhere herein, thata transgenic animal comprising a deficiency in PRCP is useful in thestudy of obesity, obesity-related disorders, and the like.

The skilled artisan will also appreciate, when armed with the presentdisclosure, that a transgenic animal comprising a deficiency in PRCP isuseful in that the transgenic mammal will comprise a lower amount ofbody fat, and is therefore a more healthful animal for humanconsumption. That is because, as disclosed herein, a transgenic animalcomprising a deficiency in PRCP is of a similar size to a counterpartanimal comprising a functional PRCP, but the transgenic animal comprisesless body fat, and is therefore a leaner animal. Further, it would beunderstood by the routineer, based upon the disclosure provided herein,that a reduction in consumption in animal fat is widely regarded as amethod to not only reduce obesity, but to maintain and improvecardiovascular health and to prevent and/or treat diabetes.

The present invention encompasses a non-human transgenic animal which iscommercially relevant as a food stuff for human consumption, as well asanimals not used as food stuffs, but where a leaner animal is of somebenefit either to the animal or to humans. Such animals include, but arenot limited to, a bird, a rodent, a cow, a pig, a sheep, a buffalo, abeefalo, a bison, a deer, a goat, and the like.

In addition, the skilled artisan would appreciate, based upon thedisclosure provided herein, that a transgenic non-human mammal lackingPRCP function is a useful model system for the study of a disease orcondition associated with, or mediated by, PRCP function, including acondition where lack of PRCP provides a benefit to the mammal. Thus, thetransgenic non-human mammal of the invention is not only useful as aleaner comestible, but also provides a useful model system for studyingthe role of PRCP function, especially as it relates to obesity,diabetes, food intake, and body weight control.

The present invention therefore includes a transgenic non-human animalcomprising a deficiency in PRCP. The skilled artisan will appreciate,when equipped with this disclosure and the data contained herein, thatan animal deficient in PRCP comprises an animal that lacks the PRCP genein its entirety, or any portion thereof, and is not limited to theportion exemplified herein.

Further, one of skill in the art will understand, when armed with thepresent disclosure, that PRCP deficiency encompasses no expression,insufficient and/or decreased expression, and/or the production of anon-functional PRCP, in that it does not exhibit the activity of PRCP asdisclosed herein.

The skilled artisan, once equipped with the teachings disclosedelsewhere herein, will readily appreciate how to produce transgenicanimals deficient in PRCP. The skilled artisan will also appreciate,while recognizing that the disclosure contained herein is in no waylimiting to the methods to produce a transgenic animal deficient inPRCP, that such animals can be produced by the deletion of the entiregene, or portions thereof. Further, the skilled artisan will appreciatethat a transgenic animal deficient in PRCP can be produced byintroducing nonsense, missense, or other mutations in the coding regionsof PRCP gene. Further, one of skill in the art will appreciate that suchanimals can be generated by mutations in the promoter/enhancer region ofthe regulatory elements governing PRCP expression. As disclosedelsewhere herein, methods to effect the expression of PRCP can alsoencompass expression of an oligonucleotide antisense to the PRCP gene,or some portion thereof, as well as the use of ribozymes and syntheticantibodies for the generation of a transgenic animal comprising adeficiency in PRCP. All such embodiments disclosed herein areencompassed in the present invention.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

For the following experiments we used NCBI Accession No. NP_(—)082519(GI 33469015), a 491 aa encoding Mus musculus angiotensinase C likeprotein and NCBI Accession No. AAH55022 (GI:32967631), a 491 aa encodingMus musculus PRCP protein. See, also Strausberg et al., Proc. Natl.Acad. Sci. U.S.A. 99(26): 16899-16903 (2002).

Obesity is a complex trait influenced by diet, energy expenditure andgenetics. Identification of genes that underlie human obesity has beendifficult, but mouse models have been used to clone and identify genessubsequently shown to contribute to human obesity. Mice provide a modelto find naturally occurring mutations that cause obesity. Mice are thebest available model, other than humans, for genetic studies of obesitybecause of their well-defined functional homology with the human genome,because obesity is influenced by hormones and neural signals from manytissues that are shared with all mammals but which are not all presentin non-mammalian models, and because of our ability to alter theirenvironment using diets that humans could eat. Further, positionalcloning can identify genes that influence phenotypes, because the genesmap coincident with the trait. Homologues and alleles of these genes canthen be tested for their effects on human physiology. Congenic modelssuch as those used here have been used to dissect the genetics ofcomplex traits.

Example 1 Identification of PRCP Gene from a Congenic Mouse Line

Material and Methods

Expression Array Analysis

Applied Biosystems Mouse Genome Survey Arrays were used to analyze thetranscriptional profiles of twenty-four RNA samples (two mice strains,three mice per tissue and four tissues-brain, gonadal white adipose,liver, and gastrocnemius) in this study. The Applied Biosystems MouseGenome Survey Array contains 32,996 60-mer oligonucleotides probesrepresenting a set of 32,381 individual mouse genes and more than 1,000control probes.

Digoxigenin-UTP labeled cRNA was generated and linearly amplified from 1μg of total RNA using Applied Biosystems Chemiluminescent RT-IVTLabeling Kit v 2.0 and manufacturer's protocol. Array hybridization,Chemiluminescence detection, image acquisition and analysis wereperformed using Applied Biosystems Chemiluminescence Detection Kit andApplied Biosystems 1700 Chemiluminescent Microarray Analyzer followingmanufacturer's protocol. Briefly, each microarray was firstpre-hybridized at 55° C. for 1 hr in hybridization buffer with blockingreagent. 18 μg of labeled cRNA targets were first fragmented byincubating with fragmentation buffer at 60° C. for 30 min, mixed withinternal control target (ICT, 24-mer oligo labeled with LIZ fluorescentdye) and hybridized to each pre-hybed microarray in a 1.5-ml volume at55° C. for 16 hr. After hybridization, the arrays were washed withhybridization wash buffer and chemiluminescence rinse buffer. Enhancedchemiluminescent signals were generated by first incubating arrays withanti-digoxigenin-Alkaline Phosphatase, enhanced with ChemiluminescenceEnhancing Solution and finally adding Chemiluminescence Substrate.Images were collected for each microarray using the 1700 analyzer.Images were auto-gridded and the chemiluminescent signals werequantified, corrected for background and spot and spatially normalized.

Statistical Analysis

For each experiment, means were compared between experimental groupsusing one-way analysis of variance (ANOVA) with mean comparisons by theStudent-Newman-Keuls Method. A level of confidence of p<0.05 was used todetermine significant differences.

Real Time PCR to Quantitate PRCP mRNA in Brain

RNA was isolated from male littermate mice derived from a cross of F1B6x B6.C-D7Mit353 mice. Whole brains were removed from mice after anovernight fast and assays performed by real time PCR in an ABI 7900.Genotypes are: “B” for background (n=8), “H” for heterozygote (n=9), and“C” for congenic or BALB/c (n=7). The assays used target three separateexons: TaqMan assay 814504 targets exon 3, TaqMan assay 814505 targetsexon 5, and TaqMan assay 1324043 targets exon 2. In addition a separateassay was performed using custom primers that were detected with Sybrgreen. Five separate control genes were used: one with Sybr Green (36B4)and four with TaqMan. Genotype effects (deltas) were determine for allprimers (Prcp and control genes). Values for the control genes wereaveraged. The exon 2 primers were calculated using a separate controlgene assay for beta-glucuronidase since this was from a separate cDNAsynthesis of the same RNA. Overall fold increased expression wascalculated as described by ABI. Results are shown in Table 5 andindicate a 1.7 fold higher expression of Prcp mRNA in lean congenic thanmore obese C57BL/6J mice.

Results

Recently, a B6.C-D7Mit353 subcongenic mouse line with less body fat thanC57BL/6J background controls has been identified (Diament et al., 2003).It is believed that the phenotypic differences (such as obesity) betweenbackground and congenic strains are due to polymorphisms in geneslocated in the congenic donor region located on chromosome 7. The leansubcongenic line contains the D7Mit353 BALB/c allele (congenic line nameB6.C-D7Mit353) on chromosome 7 and donor strain DNA from the BALB/cstrain surrounded by B6 genomic DNA for the rest of the genome. Themaximum extent of the BALB/c donor region is between D7Mit37 to D7Mit96that has the B6 alleles in the congenic. This donor region includes 97genes that may have BALC/c alleles. (Table 3).

In order to identify these genes, a whole genome oligonucleotide basedmouse microarray (ABI1700) was used to search for differentiallyexpressed genes in biological triplicates from mice homozygous for thebackground “B” and congenic “C” genotypes. mRNA expression from liver,brain, gonadal white adipose tissue and gastrocnemius muscle wereexamined since obesity may be influenced by gene expression in manytissues. RT-IVT amplified samples were hybridized to one of 29 separatemicroarrays, including duplicate hybridizations for 5 of 6 brainsamples. Chemiluminescent signals were normalized across all arraysusing quantile normalization method. Normalized data were analyzed usingthe Local Pooled Error (LPE) test of Jain et al. to identifydifferentially expressed genes. For each tissue, only genes that hadsignal/noise ratio (S/N)>=3 (i.e. 99.9% probability of the signal beingabove the background) on 2 out of 3 (4 for brain) arrays within eithermouse strain were included in the statistical analysis. Raw p-valueswere then adjusted for multiple testing by calculating false discoveryrates (FDR).

These analysis identified 330 genes passing FDR=0.05 and 188 genespassing FDR=0.01 in any one of the four examined tissues. None of thesegenes was in the congenic donor region. Seven genes in the congenicdonor region are differential in one of the tissues with raw p values<0.01, including three candidate obesity genes. Dgat2 is known toinfluence triglyceride synthesis. There is a suggestive genotype effectin muscle (see Table 1 and Table 3 where all 97 genes were mapped to thecongenic region). However, Dgat2 is unlikely to be responsible for theobserved congenic phenotype both because it does not exhibitstatistically significant regulation and because its levels areregulated opposite to those expected based on studies of Dgat2 knockoutmice. Dgat2 mRNA levels are lower in the obese background strain mice,while mice with knockouts for Dgat2 have no triglyceride (Table 1).Thyroid hormone responsive spot 14 (Thrsp) is included among the fourmost significant genes from the congenic donor region from brain. Realtime PCR for Thrsp mRNA in liver demonstrated a genotype effect that isconsistent to the liver microarray data, but which was not statisticallysignificant. The array probe for the Mgat2 (monoacylglycerol0-acyltransferase 2) gene, that is immediately next to Dgat2, did nothave detectable signals on more than 1 array for any tissues-strain.

For this reason, the focus of the subsequent studies were shifted togene prolylcarboxypeptidase (Prcp, also known as angiotensinase C orPro-x carboxypeptidase). This is because gene Prcp has previously beenassociated with the metabolic syndrome of humans (McCarthy et al., 2003)and because it is functionally related to carboxypeptidase E (Cpe) thatis the underlying cause of the “fat” mutation in mice. Tests by realtime PCR were done using four separate primers, targeting exon 2, 4 and5, and five separate endogenous control genes to rule out artefacts dueto unexpected splice variants and genotype effects on control genes.These results demonstrated statistically significant 1.77±0.33 (SD) foldhigher expression of Prcp in brains of background mice than congenicmice, 1.48±0.31 fold higher expression in heterozygous than congenicmice and 1.19±0.049 fold higher expression in background thanheterozygotes mice. These results are consistent with an additive modelwith the congenic allele promoting increased PRCP expression. To searchfor functional differences between background and congenic alleles ofPrcp, both the donor and background alleles of Prcp were sequenced. Nocoding, 3′ or 5′ untranslated region variants were found. One promoterbase change, a C→T on the donor strain was found −718 bases upstream ofthe start codon. Mouse Prcp is orthologous to humanprolylcarboxypeptidase (Prcp). Prcp in mouse is located at 81.3 Mb onchromosome 7 (Diament et al., 2003). The human ortholog is located at82.3 Mb on chromosome 11.

Example 2 α-MSH as a Substrate of PRCP

Material and Methods

Materials

α-MSH (Ac-STSMEHFRWGKPV-NH₂) (SEQ ID NO: 30) and angiotensinogen II(AgII) were purchased from Phoenix Pharmaceuticals. Inc, (Belmont,Calif.). MSH₁₋₁₂ (Ac-STSMEHFRWGKP-COOH) (SEQ ID NO: 31) was synthesizedin its purified form by the W. M. Keck Foundation Biotechnology ResourceLaboratory at Yale University. HD-Pro-Phe-Arg-paranitroanilide (S2302)was from DiaPharma (Franklin, Ohio). Human high molecular weightkininogen (HK) (18 Units/mg) and prekallikrein (PK) (21 Units/mg) wasobtained from Research Enzyme Laboratory (South Bend, Ind.).pMT/BiP/V5-His C was obtained from Invitrogen Laboratories (Carlsbad,Calif.). N-benzyloxycarbonyl-prolyl-prolinal (Z-PP) was purchased Sigma.B-PP was a kind gift of Dr. Sherwin Wilk, Mount Sinai School ofMedicine, New York.

Cloning and Expression of PRCP

pMT/BiP/V5-His C was employed as the basal plasmid in vectorconstruction. Human PRCP cDNA, lacking the first 391 base pairs of PRCP(Prcp_(nt391-1845)), was subcloned into the vector at the Kpn1 and EcoR1multiple cloning site forming pMT/Bip/V5-HisC-PRCP. The constructionmethods of the PRCP expression vector and the recombinant PRCPexpression in Schneider 2 cells followed the standard protocols(Shariat-Madar et al., 2002). The PRCP has a predicted molecular mass of51 kDa (rPRCP₅₁)

Substrate Inhibition Assay

Samples containing 8 nM rPRCP₅₁ in 100 ml total volume were prepared inthe absence or presence of increasing concentration of peptide α-MSH,MSH₁₋₁₂ or AgII in HEPES carbonate buffer (137 mM NaCl, 3 mM KCl, 12 mMNaHCO₃, 14.7 mM HEPES, 5.5 mM dextrose and 0.1% gelatin, pH 7.1containing 10 mM CaCl₂, and 1 mM MgCl₂) in microtiter plate cuvettewells with previously absorbed HK at 1 mg/well and with 20 nM PK at 37°C. The ability of purified rPRCP₅₁ to activate PK bound to HK on plasticmicrotiter plates was determined in the absence or presence ofincreasing concentrations (0.001-1 mM) of the peptide (McCarthy et al.,2003; Shariat-Madar et al., 2002). After incubation, the wells werewashed to remove unbound rPRCP and peptide and any formed kallikreinactivity was determined by addition of 100 μl of S2302 (0.8 mM) andhydrolysis was observed at 405 nm for 1 h at 37° C. The rate ofhydrolysis was recorded at different concentrations of peptide andexpressed as percent kallikrein activity remaining. Results wereexpressed as mean±SEM of 3 or more experiments.

Results

PRCP is a membrane bound protein that cleaves C-terminal amino acidslinked to a penultimate proline and can function as a protease atphysiologic pH. Thus, because of its amino acid sequence, α-MSH, acritical anorexigenic neuromodulator in the hypothalamus is a putativesubstrate of PRCP. To confirm, an in vitro assay was performed todetermine if α-MSH is a substrate inhibitor of rPRCP₅₁-inducedprekallikrein (PK) activation (FIG. 1A) (Shariat-Madar et al., 2002;2004). Recombinant PRCP₅₁ activates PK with a K_(m) and V_(max) of 9 nMand 0.2 min⁻¹, respectively. Incubation of PRCP with α-MSH at 0.001-1 mMled to a gradual decrease in the activation of PK by rPRCP₅₁ with anIC₅₀=100 mM. Likewise, angiotensinogen II (AgII), an establishedsubstrate of PRCP, inhibited PK activation with an IC₅₀=150 mM, as wasshown previously (Shariat-Madar et al., 2002; 2004; Odya et al., 1978).Alternatively, the putative product of PRCP hydrolysis of α-MSH,α-MSH₁₋₁₂, was much less effective in blocking rPRCP₅₁ activity,achieving about 20% inhibition at 1 mM (FIG. 1A). These data show thatα-MSH₁₋₁₃ with a C-terminal Pro-Val bond, but not MSH-₁₋₁₂ that lacksthe C-terminal Val, was a substrate of PRCP.

Example 3 Generation of PRCP Deficient Mice

Material and Methods

Animal Husbandry

Mice were housed and cared for according to Duhl et al. Briefly, once asubcongenic line was made, two heterozygous mice with the subcongenicregion were mated, so that their progeny had one of three genotypes.This arrangement controls for all controllable environmental variance byusing sibling mice as control mice. Mice were fed a low fat diet, weanedat 3 weeks, housed 5 per cage, and dissected at 16 weeks of age.

Preparation of PRCP Knockout Mice

ES cells (KST302) heterozygous for PRCP deletion using gene-trap vectorswere generously provided by Dr. William Skarnes, University ofCalifornia at Berkeley through BayGenomics [NHLBI-Bay Area FunctionalGenomics Consortium (http://baygenomics.ucsf.edu)] (Mountjoy et al.,1994). These PRCP gene trapped cells contain an insertion that has thefollowing regions in its vector (pGT 1-8TM) from 5′ to 3′ called SA(splice acceptor), CD4-TM, and a lacZ reporter (Skarnes et al., 1995).The KST302 cells were microinjected into C57BL/6J blastocysts at theUniversity of Michigan Transgenic Animal Model Core and surgicallyimplanted in pseudopregnant female recipients in a 129 background.Germline transmission was indicated by the presence of agouti coatcolor. Since the cloning vector contains a lacZ reporter gene, micedeleted of PRCP were screened by real time PCR using primers for LacZ(bgal) (Table 4). Genotyping of the knockout mice was performed by PCRusing DNA purified from tail biopsies with a spin column according tothe manufacturer's specifications (Promega, Madison, Wis., USA). DNAfrom wild type embryonic stem (ES) and KST302 (PRCP^(+/−) clone) cellswere used as control. The PCR conditions were 94° C. for 5 min (onecycle); 94° C. for 1 m, 60° C. for 1 m, 72° C. for 2 m (35 cycles); and72° C. for 10 min. With each run of the real time PCR, the relativeexpression of PRCP gene was normalized to that of g-actin in order toinsure fidelity between runs according to the procedure of Schmittgen etal. Animals with bgal expression on real time PCR similar to KST302cells were considered heterozygous for PRCP deletion. Animals with bgalexpression greatly increased over KST302 cells were consideredhomozygous. At the time of the present experiments, PRCP deleted micehad been backcrossed six generation into a C57BL/6 background beforehomozygous mice were prepared by breeding.

Total RNA Extraction

At time of mouse dissection, liver, spleen, brain, gastrocnemius,kidneys and four fat pads (femoral, epidydimal, retroperitoneal, andmesenteric) were removed and flash frozen in liquid nitrogen. Thesesamples were subsequently stored at −80 until RNA extraction.Approximately 100 mg of sample tissue was used for RNA extraction usingTRIzol (Life Technologies, Grand Island, N.Y.) according to themanufacturer's protocol.

Reverse Transcription

Single-stranded cDNA was synthesized using 1 μg of total RNA and TaqManreverse transcription kit (Applied Biosystems, Foster City, Calif.)according to the manufacturer's protocol.

Sequencing

Sequencing was done on cDNA for all but the 5′ UTR and 3′UTR regions ofthe genes. To sequence the 5′UTR and 3′UTR, genomic DNA was used andprimers designed based off of sequences from the Celera database.Primers were used to both amplify DNA, and later to sequence the PCRfragment after a gel extraction using QIAquick Gel Extraction Kit(Qiagen Inc., Valencia, Calif.). These primers are listed in Table 2.Primers were designed using the program Eugene v 2.2 (Daniben Inc.,Cincinnati, Ohio).

Characterization of PRCP Knockout (KO) Mice

Real time quantitative (Taqman) PCR analysis was carried out asdescribed by the vendor's instructions, with minor modifications.Briefly, measurements were performed using the iCycler iQ real-time PCRdetection system (BioRad, Hercules, Calif.). The primers (Invitrogen)and probes (ITD, Coralville, Iowa) were designed. Probes were 56-labeledwith 6-carboxyfluorescein (FAM) and a downstream 3 Black Hole quencherdye (BHQ-1) (Integrated DNA Technologies Coralville, Iowa) (Table 4).Specific primers for the real time PCR were used for the RT-PCR assayexamining insertional mutation site in kidney PRCP RNA. Melting profilesshowed the generation of specific products with melting temperatures of57.3° C. The PCR mixture (50 ml) consisted of 0.2 mM of each primer,0.02 mM probe, 1 mg RNA and TaqMan Universal Master Mix (2×) or platinumTaq. Reaction conditions were used: 95° C. for 5 min, followed by 35cycles at 95° C. for 1 m and 58 or 60° C. for 1 m. Real-time PCR datawere expressed as relative quantity based on the ratio of fluorescentchange. Negative controls (samples without polymerase) were performed inparallel during different determinations to assess melting curve andassure equivalent assay conditions. cDNA products were also analyzed forpurity by gel electrophoresis and sequencing. All assays were performedin triplicate and reported as the mean.

Sybr® Green Quantitative PCR

Prcp and control gene Arbp were also used to quantify RNA levels intissue. The control gene Arbp is a 60S acidic ribosomal protein used tonormalize loading and total RNA sample variations. The following are theSybr® Green reagent and cDNA concentrations: 1.5 μl (30 ng) of cDNA fromthe RT reaction. 2.5 μl of 3 μM forward primer, 2.5 μl of 3 μM reverseprimer, 12.5 μl of 2× Sybr Green PCR Mastermix (Applied Biosystems Inc.,Warring UK), and 6.0 μl Ambion Nuclease free water to make a 25 μlreaction volume. All primers designed to be used with Sybr® Greentechnology were created with Primer Express v 2.0.0 (Applied Biosystems,Foster City, Calif.) and the sequences of these primers are found inTable 2. To determine cDNA contamination in reagents, a no templatecontrol (NTC) replaced the 1.5 μl of cDNA with water. No reversetranscriptase reactions (−RT) controls were run to check for genomic DNAcontamination and amplification. For these reactions, 1.5 μl reversetranscriptase was replaced with 1.5 μl water. Ideally these controlsshould have C_(t) values at cycle 40 (see Data Analysis and StatisticalEvaluation), indicating no amplification. Everything was run intriplicate. Pooling of (−RT) reactions were done in order to conservereagents. No positive amplification of −RT reactions ever required us todoubt expression levels of +RT reactions. Each PCR plate was coveredwith an optical adhesive cover and inserted in ABI Prism 7900HT SequenceDetection System machine. The thermal cycling conditions were asfollows: 50° C. for 2 minutes, 95° C. for 10 minutes, 40 cycles of 95°C. for 15 seconds and 60° C. for 1 min, followed by a dissociation curveusing a gradual temperature ramping from 60° C. to 95° C. over a 5minute interval with fluorescent measurements taken every 7 to 10seconds. All reactions were performed using Model 7900HT SequenceDetector (PE Applied Biosciences, Foster City, Calif.).

Body Temperature

At approximately 15 weeks of age, anal temperature was taken. Theanimals were restrained by covering the entire mouse with a small boxthat had a slit cut out of the back of the box to allow only the tailoutside of the box. An anal temperature probe was lubricated with KYJelly and inserted approximately 1 cm into the rectum of the mouse. Theprobe was left in for approximately 10-15 seconds while the temperaturestabilized to 0.1 degrees Celsius. This procedure was done atapproximately 3 PM for three consecutive days and an average temperaturewas used for statistical analysis.

Running Wheel Activity

At approximately 8 weeks of age mice were individually housed inpolycarbonate cages equipped with electronically monitored runningwheels (Mini Mitter Co., Bend, Oreg.) cage with a running wheel andallowed free access to food and water. Running wheel data, expressed astotal revolutions per day, were collected at 15 minutes intervals usingthe VitalView Data Acquisition System (Mini Mitter, Bend, Oreg.). Micewere adjusted to the new running wheel and room environment for 2 daysbefore measurements were taken. Measurements were taken for fiveconsecutive days and calculated as “average turns per day”.

Food Intake

At 7 weeks of age, mice were individually housed in wire mesh cages, andallowed free access to food and water. Mice were adjusted to the newcage and environment for 3 days before measurements were taken. Mice andthe amount of food were weighed at the beginning of the week. At the endof seven days, the mice and food left in the cage were recorded.Spillage and feces were separated and the food spillage excluded fromthe food consumed. This procedure was repeated for the following weekwith the same mice. A one-week food intake measurement period was alsodone at 14 weeks of age. The materials and methods were the same as the7-8 weeks of age.

Spontaneous Activity

Each mouse was tested for normal exploratory locomotion. The mouse wasplaced in the photocell-equipped automated open field box. Accuscansoftware calculated total distance traversed, horizontal activity andstereotypy. The room was maintained at 22±1° C. on a 12-h light/darkcycle. Locomotor activity was measured using a Digiscan Animal ActivityMonitoring System (Omnitech Electronics, Columbus, Ohio). Mice were putin the Digiscan at 8 weeks of age with pine shavings bedding, andallowed free access to food and water. Mice were adjusted to the newroom and environment for 3 days, and measurements averaged over 4 days.

Metabolic Rate

Mice were shipped to Baylor University for metabolic rate analysis. Micewere shipped at approximately 5 weeks of age and measured atapproximately 16 weeks of age. Mice were single housed. Daytime andnight time resting energy expenditure, O₂, CO₂, heat, and weight of themouse were measured.

Data Analysis and Statistical Evaluation

Analyses of real-time quantitative PCR data were performed using thecomparative threshold cycle (C_(t)) method as suggested by AppliedBiosystems. C_(t) values are defined as the PCR amplification cycle inwhich the reporter signal is greater than the minimal detection level,and C_(t) is inversely related to the relative abundance of a particulartranscript. Only samples with C_(t) values at least five units less thanthat of the “no-RT” control were included in data analysis. To correctfor total RNA loading variations, the expression of the gene tested wasmeasured relative to the expression of control gene of Abrp.Quantification of mRNA was measured in triplicate and the Ct values wereaveraged. The relative expression of gene tested compared to Abrp wascalculated by the formula 2^([Ct(36B4)−Ct(Gene Tested)]). For ease ofunderstanding and visual presentation, the values were converted torepresent fold increase from the lowest expressed genotype such that thevalue of the lowest expressed genotype was given the arbitrary value of1.0 and all other genotypes compared to that. This calculation wasaccomplished by dividing all relative expression values by the relativeexpression value of the lowest expressing genotype. Statview (SPSS Inc.,Chicago, Ill.) was used for statistical analysis. To establish whethermRNA level differences were statistically significant for any group,one-way analysis of variance (ANOVA) was used. Significant ANOVA values(P<0.05) were followed by the Tukey-Kramer post-hoc test forsignificance to account for unequal group sizes and multiple grouptesting.

Results

Heterozygous PRCP knockout mice were prepared from KST302 cells obtainedfrom Dr. Williams Skarnes, BayGenomics. DNA from the heterozygous KST302parent cell line showed significantly more lacZ than ES cells on realtime PCR (FIG. 1B). Heterozygous PRCP KO mice were backcrossed 6generations into a C57BL6 background. Heterozygous PRCP KO mice werethen mated and their progeny examined for the presence of lacZ. Animalsproposed to be homozygous PRCP KO have significantly increased lacZexpression on real time PCR, whereas littermate controls have lacZexpression at the level of ES cells (FIG. 1B). Mice heterozygous for thePRCP deficiency have lacZ levels comparable to KST302 cells. In matingexperiments, mice presumed to be homozygous for PRCP deletion when matedwith wild type mice only produced heterozygous animals. Further, matingof heterozygous mice for PRCP deletion with wild type mice only producedheterozygous and wild type mice.

In order to determine the insertional mutation site of the gene-trappedPRCP KO mice, a sense probe from the sequence tag of this PRCP KO cellline from the BayGenomics website and an antisense probe were preparedfrom the “so-called” TM region of the cloning vector pGT1.8TM. StandardPCR was performed and the determined sequence 3′ to 5′ indicated the TMregion was preceded by the splice acceptor (SA) region of the vectorthat was preceded by the 3′ end of murine PRCP exon 5 (data not shown).These data suggested that the insertion site of the gene trap wasbetween exon and intron 5. Further studies were performed by real timeRT PCR using mRNA from KO and wild type mouse kidney, a sense probe fromexon 5, an antisense probe from the TM region of the vector, and adetection probe that spans the exon 5-SA region of the vector (Table 4).Kidney RNA of proposed KO mice, as predicted by real time PCR of lacZ,contained exon 5 and the SA-TM regions of the vector on reversetranscribed, real time PCR (FIG. 1C). Alternatively the heterozygousKST302 cells had less RNA of this insertion and kidney RNA frompredicted littermate wild type mice or ES cells had none (FIG. 1C).

Further investigations were performed to indicate that distal to thegene trap, no PRCP RNA was produced in mice predicated to be PRCP KOanimals by the DNA screening using real time PCR for lacZ (FIG. 1B).Real time RT PCR of kidney RNA from the PRCP KO and wild type mice shownin panels A and B were studied using sense probes from exons 1 or 5 andantisense probes from the vector's TM region or exons 8, 9, and 10(Table 4, FIG. 1D). The detection probe for these studies was from thecontiguous exon 5-vector SA region (Tables 4 and 5). As shown in FIG.1D, RNA was only seen in the knockout mouse between the exon 1-TMregion. No complete RNA from the KO mouse was present using probes fromexon 1 through exon 8, exon 1 through exon 10, or exon 5 through exon 9(FIG. 1D). Further, no exon 5-TM RNA was seen in the littermate wildtype mouse. These combined studies indicated that the PRCP KO mouse hasits RNA production disrupted after exon 5.

Example 4 Identification of PRCP Expression in the Brain

Material and Methods

In situ Hybridization

Mouse PRCP clone EMM1002-16394 from a kidney library prepared by the NCICGAP program (Cancer Gene Anatomy Program) was purchased from OpenBiosciences. Linearized DNA was transcribed using T7 polymerase(antisense cRNA probe) and SP6 polymerase (sense cRNA probe; RiboprobeCombination System SP6/T7, Promega Corporation, Madison, Wis.) andlabeled with ³⁵S-UTP (Amersham; 10 mCi/ml). The radiolabelled cRNA probewas then purified by passing the transcription reaction solution over aG50 column (Pharmacia Biotech) and fractions were collected and countedby using a scintillation counter. The purified cRNA probes were heatedat 80° C. for 2 minutes with 500 mg/ml yeast tRNA and 50 mM DTT in waterbefore being diluted to an activity of 5.0×10⁷ dpm/ml with hybridizationbuffer containing 50% formamide, 0.25 M sodium chloride, 1× Denhardt'ssolution and 10% dextran sulfate. Sections with this hybridizationsolution (150 ml/slide) were incubated overnight at 50° C. Followinghybridization, the slides were washed four times (10 minutes each) in4×SSC prior to RNase digestion (20 mg/ml for 30 min at 37° C. ), rinsedat room temperature in decreasing concentrations of SSC that contained 1mM DTT (2×, 1×, 0.5×; 10 minutes each) to a final stringency of 0.1×SSCat 65° C. for 30 minutes (20). After dehydration in increasing alcohols,the sections were exposed to b-max hyperfilm (Amersham) for 5 daysbefore being dipped in Kodak NTB-2 liquid emulsion diluted 1:1 withdistilled water. The dipped autoradiograms were developed 21 days laterwith Kodak D-19 developer, fixed, and the sections counterstainedthrough the emulsion with hematoxylin. Sections were examined underbrightfield and darkfield illumination. Several control experiments werecarried out to test the specificity of the hybridization method and DIIprobe. First, sections were incubated as described above withhybridization solution containing the sense-strand probe synthesized byusing SP6 polymerase to transcribe the coding strand of the DNA insert.Second, the hybridization was also attempted on sections that had beenpretreated with RNase (20 mg/ml for 30 min at 37° C.) to degrade tissueRNA. Third, tissue sections were incubated in radiolabelled probe andthen in an excess of unlabeled probe, which competed with theradiolabelled probe, eliminating the increased signal. Moreover, toassess the thermal stability of the hybrid, different series of sectionswere rinsed in 0.1×SSC at 75° C., 80° C., 85° C., 90° C., 94° C., and98° C.

X-Gal Staining and Immunocytochemistry

PRCP KO mice were perfused with 4% paraformaldehyde. Brains weresectioned with vibratome (40 μm) and washed with PBS (137 mM NaCl, 2.7mM KCl, 8 mM Na₂HPO₄, 2.6 mM KH₂PO₄) 4 times. Sections were then rinsedquickly once in cold PBS plus 2 mM MgCl₂ and incubated in the abovesolution for 10 min at 4° C. Permeabilization was performed byincubation in cold PBS with detergent (0.01% sodium desoxycholate and0.02% NP40) for 10 min. Sections were then incubated overnight at 37° C.in the staining solution containing 25 mM K₃Fe(CN)₆, 25 mMK₄Fe(CN)₆3H2O, 2 mM MgCl2 in PBS and 1 mg/ml of X-Gal. Sections werethen rinsed in PBS and processed for immunocytochemistry. The followingantisera were used: mouse b-endorphin, (diluted 1: 5000; Chemicon,Temecula, Calif.) rabbit anti-MCH (diluted 1:12000; PhoenixPharmaceutics, Belmont, Calif.), goat anti-Hcrt b (diluted 1:3000; SantaCrus Biotechnology, Santa Cruz, Calif.) and mouse anti-thyrosinehydroxylase (TH; diluted 1:5000; Sigma). Antisera were incubatedovernight at room temperature. After several washes with PB, sectionswere incubated in their appropriate secondary antibody (biotinylatedgoat anti rabbit, horse anti mouse, horse anti goat IgG; 1:250 in PB;Vector Laboratories, Inc., Burlingame, Calif.) for 2 h at roomtemperature, then rinsed in PB three times 10 min each time, andincubated for 2 h at room temperature with avidin-biotin-peroxidase(ABC, 1:50 in PB; ABC Elite Kit, Vector Laboratories, Inc.). Theimmunolabeling was visualized with the nickel-diaminobenzidine (DAB)reaction (15 mg DAB, 165 μl 0.3% H₂O₂ in 30 ml PB) for 5-10 min at roomtemperature resulting in a light brown reaction product.

Results

To clarify the phenotype of PRCP-expressing cells in the hypothalamus,beta galactosidase (β-Gal) expression in hypothalamic slices in PRCP KOmice was analyzed. Expression of β-Gal in the central nervous system wasfound abundant in various regions, including the cerebral cortex, brainsstem, hippocampus and hypothalamus. In the hypothalamus, β-Gal labelledcells were highly expressed in the lateral hypothalamus(LH)-perifornical area and zona incerta (ZI) (FIG. 2A). Labeled cellswere also found in the arcuate nucleus, as well as in the dorsalmedialnucleus of the hypothalamus (FIG. 2A). The expression of β-Gal in PRCPKO animals was virtually identical to the distribution pattern of PRCPmRNA in wild type animals as revealed by in situ hybridization of PRCPmRNA (FIG. 2B).

In the arcuate nucleus, the majority of POMC-immunolabeled cells lackedβ-Gal expression (FIG. 2) suggesting that if PRCP cleaves α-MSH, itshould occur at α-MSH release sites. In hypothalamic regions where PRCPis most abundantly expressed, lateral hypothalamus-perifornical regionand dorsomedial nucleus, there are two known orexigenic peptidergiccircuits that project to α-MSH -targeted sites: one producing melaninconcentrating hormone (MCH) and the other hypocretin/orexin (Hcrt)(Vaughan et al., 1989; Skofitsch et al., 1985; Bittencourt et al., 1992;De Lecea et al., 1998; Sakurai et al., 1998; Trivedi et al., 1998;Horvath et al., 1998; 1999). Double labelling studies revealed thatpopulations of MCH and Hcrt neurons also expressed β-Gal (FIG. 2). Axonterminals of both of these systems, as well as, occasionalβ-Gal-labelled boutons were found in direct apposition toα-MSH-containing axon terminals in various parts of the hypothalamus,including the paraventricular nucleus (FIG. 2). These observationsrevealed that hypothalamic PRCP cells are in ideal anatomical positionto affect α-MSH action.

Example 5 Effect of α-MSH to α-MSH₁₋₁₂ on Food Intake

Material and Methods

ICV Injection of MSH and MSH_(1-12:)

Eighteen male rats (200-250 gr) were used in this study. Animals with athird ventricle cannulation were purchased from Taconic Farms Inc. Ratswere single caged and divided in 3 groups: animals injected with saline,animals injected with 2.5 μg of □-MSH and animals injected with 2.5 μgof α-MSH₁₋₁₂. All the rats were fasted overnight. The next morning, theanimals were injected and a known amount of food was given and measuredafter 2 hours from re-feeding (Abbott et al., 2000).

Electrophysiology

Coronal hypothalamic brain slices (˜300 μm) were prepared with aVibratome from POMC-GFP transgenic mice. Slices were maintained at roomtemperature in artificial cerebrospinal fluid saturated with 95% O2 and5% CO2. Slices were equilibrated in the recording chamber for 1 hr at35° C. in ACSF saturated with 95% O2 and 5% CO2 prior to recordings.ACSF contains (mM): NaCl, 124; KCl, 3; CaCl2, 2; MgCl2, 2; NaH2PO4,1.23; NaHCO3, 26; glucose, 10; pH 7.4 with NaOH. An uprightfluorescence/infrared microscope with long working distance objectives(Olympus BX51WI) and fluorescence filter sets for GFP (Chroma TechnologyCorp) was used. Fluorescent POMC-GFP neurons were selected and a patchpipette (4-6 mega Ohm resistance) was advanced onto the surface of theneuron by a micromanipulator (Sutter 225). The pipette solution contains(in mM) K gluconate, 128; HEPES, 10; EGTA, 1; KCl, 10; MgCl₂, 1; CaCl₂,0.3; (Mg)-ATP, 5; (Na)GTP, 0.3, PH.7.4. Whole cell patch clampexperiments were performed with a Multiclamp 700A amplifier (AxonInstruments, Inc). Whole cell current clamp was used to monitor theeffect of α-MSH on membrane potential of recorded neurons held atresting membrane potential. All data were sampled at 3-10 kHz andfiltered at 1-3 kHz with an Apple Macintosh computer using Axograph 4.9(Axon Instruments). Eletrophysiological data were analyzed with Axograph4.9 (Axon Instruments), plotted with Igor Pro software (WaveMetrics,Lake Oswego, Oreg., USA) and presented as the mean±SEM. Statisticalanalysis was performed by unpaired T test. The level of statisticalsignificance was set at P<0.05.

Results

To examine whether conversion of α-MSH to α-MSH₁₋₁₂ by PRCP could affectfood intake, the effect of intracerebroventricular administration ofα-MSH and α-MSH₁₋₁₂ on food intake in fasted animals was compared(Abbott et al., 2000). In 12 hour-fasted animals, 2.5 μg of α-MSHinduced a 40% reduction in food intake (5.21±0.50 g) compared to thesaline-treated animals (8.71±0.56 g; FIG. 3A). On the other hand, 2.5 μgof α-MSH₁₋₁₂ did not significantly affect food intake (8.89±0.90 g)compared to the saline controls (8.71±0.56 gr; FIG. 3A).

The lack of suppression of food intake by α-MSH₁₋₁₂ suggests that thehydrolysis product of α-MSH by PRCP is less active in regulatingneuronal functions via melanocortin receptors. To test for that,electrophysiological responses of POMC perikarya to α-MSH and α-MSH₁₋₁₂was analyzed using patch-clamp in vitro slice recordings from transgenicanimals expressing green fluorescence protein (GFP) driven by the mousePOMC promoter (Pinto et al., 2004). This analysis is based on thepreviously tested phenomenon that activation of MC3 autoreceptorsinduces hyperpolarization of POMC perikaryal membrane potentials (Cowleyet al., 2001). Bath application of α-MSH induced hyperpolarization(66-mV vs. 46-mV) of POMC membrane potential, an effect that wasundetectable after application of α-MSH₁₋₁₂ (FIG. 3B). Thiselectrophysiological analyzes confirmed that the hydrolysis product ofα-MSH by PRCP is ineffective in triggering adequate neuronal responses.

Example 6 Inhibition of PRCP

Material and Methods

ICV Injection of B-PP

Twelve male rats (200-250 gr) were used in this study. Animals with athird ventricle cannulation were purchased from Taconic Farms Inc. Ratswere single caged and divided in 2 groups: animals injected with 0.9 μgof B-PP and animals injected with the same volume of vehicle. A knownamount of food was given to the animals and weighted every 24 hours.

IP Injection of B-PP

For this study 10 ob/ob mice were used. Mice were injected at thebeginning of the dark phase intraperitoneally with 2 doses of 400 pg ofB-PP every 24 hours and food intake was measured before each injectionand every 24 hours after the last administration. Control mice wereinjected at the same times and with same volume of vehicle.

IP Injection of Z-PP

For this study 10 ob/ob mice were used. Mice were injected at thebeginning of the dark phase intraperitoneally with 2 doses of 5 mg Z-PPevery 12 hours and food intake was measured every 12 hours after thelast injection. Control mice were injected at the same times and withthe same volume of vehicle. Note that for Z-PP doses lower than 5 mgwere ineffective in reducing food intake. Moreover, the lasting effectwas also reduced (after 24 hours from the last injection there was nodifference in food intake compared to vehicle controls).

IP Administration of α-MSH and MTII to PRCP KO and Wild Type Mice

Wild type and PRCP KO mice were used in this study. Animals were singlecaged and injected with saline at the beginning of the dark phase for 3days. For each mouse, food intake was measured at 4, 12 and 24 hoursafter the injection. After 3 days, animals were divided in 6 groups: 1)wild type mice receiving 200 nmol MTII in saline; 2) wild type micereceiving 200 nmol α-MSH in saline; 3) wild type mice receiving saline;4) PRCP KO mice receiving 200 nmol of MTII in saline; 5) PRCP KO micereceiving 200 nmol α-MSH; 6) PRCP KO mice receiving saline.

Results

If PRCP-regulated hydrolysis of α-MSH has a physiological role inregulation of food intake, then inhibition of PRCP enzymatic activityshould interfere with feeding behaviour. To test this, the effects oftwo previously characterized inhibitors of PRCP, t-Butyl Carbamate(BOC)-prolyl prolinal (B-PP) and N-benzyloxycarbonyl-prolyl-prolinal(Z-PP) (Wilk et al., 1983; Friedman et al., 1984) was analyzed on foodintake. Intracerebroventricular administration of B-PP (0.9 mg) to ratsinduced suppression of overnight food intake (36.7% versus 94.91% ofcontrols after 24 hours from the administration), which was followed byrecovery to controls levels without detectable rebound feeding (89.24%versus 97.67% of controls after 48 hours from the administration (FIG.3C). After administration of B-PP systemically (400 μg) to obese, leptindeficient (ob/ob) mice it was found that B-PP suppressed food intake andbody weight gain of these genetically obese animals (FIG. 3D). The otherinhibitor of PRCP, Z-PP, was also tested in ob/ob animals (5 mg ip) andwas found to suppress food intake, but with lower efficacy than that ofB-PP (FIG. 3E). Nevertheless, these observations showed that targetingPRCP activity with central or peripheral administration of proteaseinhibitors can reduce food intake and body weight gain.

Despite of α-MSH's well described anorectic function and the recognitionof MC4R as critical mediator of melanocortins' food reducing effects(Fan et al., 1997), peripheral administration of α-MSH has not beenreported to reduce food intake. In contrast, a synthetic analog ofmelanocortins, MTII (Ac-Nle⁴-c[Asp⁵, D-Phe⁷, Lys¹⁰] α-MSH-(4-10)-NH₂) isreadily able to reduce food intake even after peripheral administration(Fan et al., 1997). The diminished efficacy of peripheral α-MSH on foodintake together with the recent discovery of α-MSH variants that aremore stable Guo et al., 1997), suggest that extracellular degradation ofα-MSH is an important element in determining its anorexigenic efficacy.To test whether PRCP masks the anorectic effects of peripheral α-MSHadministration, the effect of peripherally administered α-MSH on foodintake regulation in wild type and PRCP KO mice were tested and thevalues were compared to those obtained by peripheral MTIIadministration. Intraperitoneal injection of α-MSH (200 nmol) into wildtype mice had no effect on feeding (FIG. 4), while MTII (200 nmol)administration resulted in significant decline in food intake (35.5%food intake compared to 100% controls; FIG. 4). However, ip. injectionof α-MSH (200 nmol) into PRCP-ablated mice strikingly had an equivalenteffect of food intake reduction as MTII (34.3% food intake compared 100%controls; FIG. 4). Because PRCP is expressed in various peripheraltissues as well as in the brain, it is reasonable to suggest that PRCPis responsible for inactivation of peripherally injected α-MSH regardingfood intake regulation.

In summary, the B6.C-D7MIt353 congenic with a maximum of 14.7 megabasesof BALB/cJ donor DNA on a C57BL/6J background (see Table 3) includes atleast one statistically significant functional obesity candidategene-Prcp. Survey of all other genes in the donor region using amicroarray with complete coverage revealed no other genes withstatistically significant differential expression. The discovery thatPRCP is a positional candidate obesity gene, despite its being locatedin a haplotype shared by B6 and the BALB/c donor strain, motivatedmechanistic studies which have now shown that it is a member of avalidated human obesity pathway. The data disclosed herein demonstratethat pharmacological inhibition of PRCP could be used to successfullycombat obesity and related disorders.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom as modifications will be obvious to those skilled in the art.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

REFERENCE

The disclosures of each and every patent, patent application, andpublication cited herein including but limited to the references listedimmediately below are hereby incorporated herein by reference in theirentirety.

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TABLE 1 Congenic Region Genes with LPE pval <0.01

TABLE 2 Prcp sequencing primers and quantitative PCR primers Prcp1upstreamproxf: SEQ ID Sequencing 5′-CAAAGGACACAGACAAGCCC-3′ NO: 131upstreamproxr: SEQ ID 5′-CCACACTCAACCAGAACAGC-3′ NO: 14 2upstreamproxf:SEQ ID 5′-GTCCAACTCCGTCTGGAAGG-3′ NO: 15 2upstreamproxr: SEQ ID5′-AAGACAGGAGCAGGAGAGCC-3′ NO: 16 5′UTRproxf: SEQ ID5′-TGCCTGGTTTTAGGTTTCCC-3′ NO: 17 5′UTRproxr: SEQ ID5′-AAGACAGGAGCAGGAGAGCC-3′ NO: 16 1proxf: SEQ ID5′-CTTTAGTCGCCCACACTGTG-3′ NO: 18 1proxr: SEQ ID5′-CAGGTTCACCCAGGTCTCAG-3′ NO: 19 2proxf: SEQ ID5′-CAGCCTGGTTCAGGATGAAG-3′ NO: 20 2proxr: SEQ ID5′-CACAGAGGAAGGATCGAAGG-3′ NO: 21 3proxf: SEQ ID5′-CTATCAACATCCATGACGGGGC-3′ NO: 22 3proxr: SEQ ID5′-TTGGCAGAATGTAGCCAGAC-3′ NO: 23 3′UTRproxf: SEQ ID5′-GCCTTGTCCTGCTCTCTTTG-3′ NO: 24 3′UTRproxr: SEQ ID5′-AAGTCCCCACACACCTGATG-3′ NO: 25 Sybr Primers prcpabif: SEQ ID for Prcp5′-GAACTACCCTTACGCATGCAACT-3′ NO: 26 prcpabir: SEQ ID5′-AATATTGGCACACCTCCTTGATG-3′ NO: 27 Sybr primers AbrpF: SEQ ID for Abrp5′-GGACCCGAGAAGAGCTCCTCCTT-3′ NO: 28 AbrpR: SEQ ID5′-TCAATGGTGCCTCTGGAGATT-3′ NO: 29

TABLE 3 Panther Function LocusLink RefSeq Cyto Start Loc Congenic OrderAK014876 84183929 1 67669 NM_026304 7 D3 84236719 2 Transcription factor-> Other 13626 NM_021876 7 47.0 cM     3 3 transcription factor AK04604584351921 4 Transcription factor -> Basal 84432318 5 transcription factorAK019082 84449997 6 Membrane traffic protein -> 233489 NM_146194 7 47.2cM 84455387 7 Vesicle coat protein Membrane traffic protein -> 83671 NM031394 7 D3 84629289 8 Membrane traffic regulatory protein AK00610584753586 9 233490 7 D3 84770337 10 66271 84778195 11 BC049680 8479573312 AK046356 84804627 13 Cytoskeletal protein -> Actin 23859 NM 011807 7D3 85763169 14 family cytoskeletal protein Cell junction protein ->Other cell junction protein 86855206 15 73845 NM 028665 7 D3 86881740 16Nucleic acid binding -> NM 029078 86934924 17 Nuclease; Nucleic acidbinding -> MrnA processing factor Select regulatory molecule -> 75985 NM029494 7 D3 87932620 18 G-protein -> Small GTPase NM 028729 87141999 19Protease -> Serine protease 72461 NM 028243 7 D3 87163575 20 87219883 2158238 NM 021427 7 D3 87289017 22 88313547 23 Protease -> Other proteasesAK034106 88655262 24 Viral protein-> Other viral protein 88655243 25Nucleic acid binding -> Other 89737851 26 RNA-binding protein 66365 NM025515 7 91033224 27 AK077305 91010506 28 Nucleic acid binding -> 15012NM 008251 16 69.8 cM 94669420 29 Chromatin/chromatin-binding proteinTransferase 381903 NM_199035 7 E1 94676128 30 Oxidoreductase 68197NM_024220 7 E1 94711155 31 21835 NM_009381 7 E1 94723941 32 BC02458894764257 33 101861 NM_027256 7 E1 94793837 34 66273 NM_183251 7 E194865537 35 BC054454 94898008 36 AK005667 94916464 37 AK041721 9496980538 Transporter -> Other 12729 NM_023671 7 50.0 cM 95016473 39transporter 66333 NM_175105 7 E1 95046310 40 Kinase -> Protein kinase ->18479 AK078711 7 46.5 cM 95110313 41 Non-receptor serine/threonineprotein kinase 233537 NM_177696 7 E1 95241025 42 Cytoskeletal protein ->Actin 17921 NM_008663 7 48.1 Cm 95371537 43 family cytoskeletal protein-> Actin binding motor protein Protease -> Cysteine 12337 7E1 9544227144 proteaseSelect calcium binding protein -> Calmodulin related proteinSignaling molecule -> Other 18378 NM_011010 7 48.0 cM 95465226 45signaling molecule 330599 NM_177896 7E1 95498522 46 Transferase ->BC039789 95514484 47 Glycosyltransferase AK053951 95527630 48 ReceptorAK035461 95671996 49 Oxidoreductase BC059730 #REF! 50 Lyase -> Cyclase-> 95766435 51 Guanylate cyclase Defense/immunity protein -> 95817397 52Antibacterial response protein 233545 NM_172280 7E1 95908991 53 AK05290996006624 54 72981 7F1 96024131 55 Signaling molecule -> Other 22411NM_009519 7 48.0 cM 96156716 56 signaling molecule 78610 AK020590 7E196208474 57 Transferase -> 67800 NM_026384 7E1 96478091 58Acyltransferase Transferase -> 233549 NM_177448 7E1 96544118 59Acyltransferase BC050000 96586291 60 17760 NM_010837 7 E2-F1 96593839 61ChaperoneSelect regulatory 12406 NM_009825 7E1 62 molecule -> Proteaseinhibitor -> Serine protease inhibitor 233552 96706470 63 AK01301296790689 64 Nucleic acid binding -> 27050 7 49.6 cM 96802892 65Ribosomal protein Miscellaneous function -> 109689 NM_178220 7 50.0 cM96859831 66 Other miscellaneous function protein Cell adhesion moleculeAK032609 96950950 67 Transporter -> Other 101488 NM_175316 7E1 9698370368 transporter Receptor -> G-protein coupled 259066 NM_147063 9705809269 receptor Receptor -> G-protein coupled 258353 258353 97090113 70receptor Hydrolase -> Other hydrolase 50877 NM_016720 97136643 71Protease 66624 7 E1 97163797 72 AK040058 97197602 73 97201983 7497252039 75 Ligase -> Other ligase 67164 7 E1 97493860 76 Ion channel ->Voltage-gated 57442 7 E1 97513242 77 ion channel -> Voltage-gatedpotassium channel Isomerase -> Mutase 70974 NM_027629 7 E1 97562568 78Oxidoreductase -> 320452 NM_177161 7 E1 97621543 79 HydroxylaseHydrolase -> Esterase 72590 NM_028292 7 E1 97664564 80 BC046540 9770940281 97763713 82 Chaperone -> Other 69387 NM_153527 7 E1 97839315 83chaperones 68185 NM_183270 7 E1 97875299 84 52443 NM_198831 4 42.5 cM97887139 85 Select regulatory molecule -> 19346 NM_024287 7 E1 9794656486 G-protein -> Small GTPase Membrane traffic protein -> 27276 NM_0137467 E1 97983081 87 Other membrane traffic protein Receptor -> Cytokinereceptor 320100 NM_177073 7 E1 98196483 88 -> Tumor necrosis factorreceptor BC028887 98220370 89 Receptor -> G-protein coupled 233571NM_18316 7 E1 98288704 90 receptor 207278 NM_199012 7 E1 98462084 91Nucleic acid binding -> 98507948 92 Ribosomal protein BC060054 9864569893 Transfer/carrier protein -> 56018 7 E1 98675709 94 Othertransfer/carrier protein 69710 NM_027180 98706605 95 Hydrolase ->BC006845 98780598 96 Phosphodiesterase Transferase -> 11871 NM_007490 749.0 cM 98916271 97 Glycosyltransferase

TABLE 4 Primers for the Characterization of the PRCP KO Mice PositionPosition Position On On On Vector Vector Vector Target Sense Primer orcDNA Antisense primer or cDNA Probe # or cDNA Geno- typing B-lac Z5′-ATGGCGATTACCGTTGATGT-3′ 5146- 5′- 5247- 5′-56-FAM/ 5167-5185 (SEQ IDNO: 2) 5165 CCAGTTTACCCGCTCTGCTA- 5228 GAAGTGGCGAGCGATACAC/ 3′ 3BHQ1/-3′(SEQ ID NO: 3) (SEQ ID NO: 4) Inser- tional mutation site E5-TM5′-GGCATGCTTGCAGCCTGGTT-3′  606-626 5′- 1952- 5′-56-FAM/  621-630: (SEQID NO: 5) GTGATCCAGGACTGGGAAGA- 1932 TTGTAGTTGGGTCCCAGGTC/ 1715-1725 3′3BHQ1/-3′ (SEQ ID NO: 6) (SEQ ID NO: 7) PRCP mRNA E1-TM5′-TGCTGGTGGCTCTCTTTCT-3′   16-34 5′- 1952- 5′-56-FAM/  621-630: (SEQ IDNO: 8) GTGATCCAGGACTGGGAAGA- 1932 TTGTAGTTGGGTCCCAGGTC/ 1715-1725 3′3BHQ1/-3′ (SEQ ID NO: 6) (SEQ ID NO: 7) E1-E8 3′-TGCTGGTGGCTCTCTTTCT-3′  16-34 5′- 1150- 5′-56-FAM/  621-630: (SEQ ID NO: 8)AAAGGGCATGACCATTTCTG- 1131 TTGTAGTTGGGTCCCAGGTC/ 1715-1725 3′ 3BHQ1/-3′(SEQ ID NO: 9) (SEQ ID NO: 7) E1-E10 5′-TGCTGGTGGCTCTCTTTCT-3′   16-345′- 1333- 5′-56-FAM/  621-630: (SEQ ID NO: 8) CCACGGGTCTAACTCACCAT- 1314TTGTAGTTGGGTCCCAGGTC/ 1715-1725 3′ 3BHQ1/-3′ (SEQ ID NO: 10) (SEQ ID NO:7) E5-E9 5′-  496-520 5′- 1195- 5′-56-FAM/  621-630:TTTTGCAGAGTTAATCAGACACTTG- GTCCCACAGAAAGGGTTCAA- 1176TTGTAGTTGGGTCCCAGGTC/ 1715-1725 3′ 3′ 3BHQ1/-3′ (SEQ ID NO: 11) (SEQ IDNO: 12) (SEQ ID NO: 7) Genotyping by real time PCR was performed withtail DNA with the indicated primers. The characterization of theinsertional mutation site was performed with mRNA from a wild type andKO mouse. “E” stands for exon region on PRCP; “TM” stands for the TMregion on trapping vector pGT1.8TM. * indicates two numbering locations:the smaller numbers are the location of the 3′ end of exon 5 from PRCPcDNA; the larger numbers indicate the numbering of the SA region on thevector pGT1.8TM. These regions are contiguous in the PRCP gene ofgene-trapped mice.

TABLE 5 Delta Delta (Prcp - Control) Ct Genotype (C − B) - (C − H) - (H− B) - values effects Average fold Average fold Average fold B C H C − BC − H H − B control B/C control H/C control B/H Prcp by Sybr green 27.8128.82 28.66 1.01 0.16 0.85 0.97 1.95 0.66 1.58 0.31 1.24 Exon 2 31.5632.14 31.50 0.58 0.64 −0.06 1.05 2.07 0.65 1.57 0.40 1.32 Exon 3 30.1530.97 30.84 0.82 0.13 0.69 0.78 1.71 0.63 1.55 0.15 1.11 Exon 5 29.4930.26 30.35 0.77 −0.09 0.86 0.73 1.65 0.41 1.33 0.32 1.24 36B4 by Sybr20.39 20.30 20.93 −0.09 −0.63 0.54 Beta-Glucuronidase 27.97 28.12 28.530.15 −0.41 0.56 Beta-2-microglobulin 22.95 22.81 23.48 −0.14 −0.67 0.53TBP 26.23 26.35 26.95 0.12 −0.60 0.72 Glyceraldehyde 20.73 20.91 21.100.18 −0.19 0.37 phosphate dehydrogenase Beta-2-microglobulin 29.12 28.6528.66 −0.47 −0.01 −0.46 (assay for Exon 2) Exon 2 = ABI TaqMan assaynumber 1324043 Exon 3 = ABI assay number 804504 Exon 5 = ABI assaynumber 804505

1. A method of preventing or treating obesity and obesity-relateddisorders in an animal in need thereof, said method comprisingadministering to the animal a prolylcarboxypeptidase (PRCP) inhibitingamount of a PRCP inhibitor.
 2. The method of claim 1, wherein said PRCPinhibitor is selected from the group consisting of a ribozyme, a siRNA,an antisense nucleic acid, an aptamer, an antibody, a peptide and achemical compound.
 3. The method of claim 2, wherein said chemicalcompound is selected from the group consisting of t-butyl carbamate(BOC)-prolyl prolinal (B-PP), N-benzyloxycarbonyl-prolyl-prolinal(Z-PP), diisopropyl fluorophosphate, PMSF, antipain, leupeptin, corntrypsin, and mercuric chloride.
 4. The method of claim 3, wherein saidchemical compound is selected from the group consisting of B-PP andZ-PP.
 5. The method of claim 2, wherein said peptide is selected fromthe group consisting of α-melanocyte stimulating hormone (α-MSH) andangiotensinogin II (AgII).
 6. The method of claim 1, wherein said PRCPinhibitor is a PRCP substrate.
 7. The method of claim 1, wherein theanimal suffers from or is predisposed to obesity.
 8. The method of claim1, wherein the animal is obese and suffers from or is predisposed to oneor more obesity-related disorders selected from the group consisting ofhypertension, dyslipidemia, diabetes, stroke, gallbladder disease,cardiovascular disease, osteoarthritis, rheumatoid arthritis,hypercholesterolemia, stable angina, unstable angina, sleep apnea,respiratory problems, cancer, stroke hyerinsulinemia, Syndrome X,hypercholesterolemia, hyperlipoproteinernia, hypertriglyceridemia,atherosclerosis, diabetic renal disease.
 9. The method of claim 8,wherein the disorder is diabetes.
 10. A method for inducing weight loss,decreasing body fat and/or decreasing food intake in an animal in needthereof, said method comprising administering to the animal a PRCPinhibiting amount of a PRCP inhibitor, thereby inducing weight loss,decreasing body fat, and/or decreasing food intake in said animal. 11.(canceled)
 12. The method of claim 10, wherein said animal is selectedfrom the group consisting of a bird, a rodent, and a mammal, and furtherwherein said mammal is selected from the group consisting of a cow, apig, a sheep, a buffalo, a beefalo, a bison, a deer, a goat, and ahuman.
 13. (canceled)
 14. A method for affecting appetite. affectingphysical activity, and/or affecting metabolic rate in an animal in needthereof, said method comprising administering a PRCP inhibiting amountof a PRCP inhibitor to the animal, thereby affecting appetite, affectingphysical activity and/or affecting metabolic rate in said animal. 15-16.(canceled)
 17. The method of claim 1, wherein the PRCP inhibitor isadministrated peripherally.
 18. The method of claim 1, wherein the PRCPinhibitor is administrated centrally. 19-26. (canceled)